Human fatty acid beta-oxidation enzymes

ABSTRACT

The invention provides human fatty acid beta-oxidation enzymes (HUFA) and polynucleotides which identify and encode HUFA. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for treating or preventing disorders associated with expression of HUFA.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of humanfatty acid beta-oxidation enzymes and to the use of these sequences inthe diagnosis, treatment, and prevention of genetic disorders, neuronaldisorders, cancer, infectious diseases, liver disorders, and cardiac andskeletal muscle disorders.

BACKGROUND OF THE INVENTION

Mitochondrial and peroxisomal beta-oxidation enzymes degrade saturatedand unsaturated fatty acids by sequential removal of two-carbon unitsfrom Coenzyme A (CoA)-activated fatty acids. The main beta-oxidationpathway degrades both saturated and unsaturated fatty acids while theauxiliary pathway performs additional steps required for the degradationof unsaturated fatty acids.

The pathways of mitchondrial and peroxisomal beta-oxidation use similarenzymes, but have different substrate specificities and functions.Mitochondria oxidize short-, medium-, and long-chain fatty acids toproduce energy for cells. Mitochondrial beta-oxidation is a major energysource for cardiac and skeletal muscle. In liver, it provides ketonebodies to the peripheral circulation when glucose levels are low as instarvation, endurance exercise, and diabetes. (Eaton, S. et al. (1996)Biochem. J. 320:345-357.) Peroxisomes oxidize medium-, long-, andvery-long-chain fatty acids, dicarboxylic fatty acids, branched fattyacids, prostaglandins, xenobiotics, and bile acid intermediates. Thechief roles of peroxisomal beta-oxidation are to shorten toxiclipophilic carboxylic acids to facilitate their excretion and to shortenvery-long-chain fatty acids prior to mitochondrial beta-oxidation.(Mannaerts, G. P. and Van Veldhoven, P. P. (1993) Biochimie 75:147-158.)

The auxiliary beta-oxidation enzyme 2,4-dienoyl-CoA reductase catalyzesthe following reaction:

    trans-2, cis/trans-4-dienoyl-CoA+NADPH+H.sup.+ trans-3-enoyl-CoA+NADP.sup.+

This reaction removes even-numbered double bonds from unsaturated fattyacids prior to their entry into the main beta-oxidation pathway.(Koivuranta, K. T. et al. (1994) Biochem. J. 304:787-792.) The enzymemay also remove odd-numbered double bonds from unsaturated fatty acids.(Smeland, T. E. et al. (1992) Proc. Natl. Acad. Sci. USA 89:6673-6677.)

Rat 2,4-dienoyl-CoA reductase is located in both mitochondria andperoxisomes. (Dommes, V. et al. (1981) J. Biol. Chem. 256:8259-8262.)Two immunologically different forms of rat mitochondrial enzyme existwith molecular masses of 60 kDa and 120 kDa. (Hakkola, E. H. andHiltunen, J. K. (1993) Eur. J. Biochem. 215:199-204.) The 120 kDamitochondrial rat enzyme is synthesized as a 335 amino acid precursorwith a 29 amino acid N-terminal leader peptide which is cleaved to formthe mature enzyme. (Hirose, A. et al. (1990) Biochim. Biophys. Acta1049:346-349.) A human mitochondrial enzyme 83% similar to rat enzyme issynthesized as a 335 amino acid residue precursor with a 19 amino acidN-terminal leader peptide. (Koivuranta, supra.) These cloned human andrat mitochondrial enzymes function as homotetramers. (Koivuranta,supra.) A Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA reductaseis 295 amino acids long, contains a C-terminal peroxisomal targetingsignal, and functions as a homodimer. (Coe, J. G. S. et al. (1994) Mol.Gen. Genet. 244:661-672; and Gurvitz, A. et al. (1997) J. Biol. Chem.272:22140-22147.) All 2,4-dienoyl-CoA reductases have a fairly wellconserved NADPH binding site motif of sequence-h-X-h-X-Gly-X-Gly-X-X-Gly-X-X-X-h-X-X-h- . . . Asp/Glu-, whereh=hydrophobic amino acid residue and X=any amino acid residue.(Koivuranta, supra.)

The main pathway beta-oxidation enzyme enoyl-CoA hydratase catalyzes thefollowing reaction:

    2-trans-enoyl-CoA+H.sub.2 O+3-hydroxyacyl-CoA

This reaction hydrates the double bond between C-2 and C-3 of2-trans-enoyl-CoA, which is generated from saturated and unsaturatedfatty acids. (Engel, C. K. et al. (1996) EMBO J. 15:5135-5145.) Thisstep is downstream from the step catalyzed by 2,4-dienoyl-reductase.Different enoyl-CoA hydratases act on short-, medium-, and long-chainfatty acids. (Eaton, supra.) Mitochondrial and peroxisomal enoyl-CoAhydratases occur as both mono-functional enzymes and as part ofmulti-functional enzyme complexes. Human liver mitochondrial short-chainenoyl-CoA hydratase is synthesized as a 290 amino acid precursor with a29 amino acid N-terminal leader peptide. (Kanazawa, M. et al. (1993)Enzyme Protein 47:9-13; and Janssen, U. et al. (1997) Genomics40:470-475.) Rat short-chain enoyl-CoA hydratase is 87% identical to thehuman sequence in the mature region of the protein and functions as ahomohexamer. (Kanazawa, supra; and Engel, supra) A mitochondrialtrifunctional protein exists that has long-chain enoyl-CoA hydratase,3-hydroxyacyl-CoA dehydrogenase, and long-chain 3-oxothiolaseactivities. (Eaton, supra.) In human peroxisomes, enoyl-CoA hydrataseactivity is found in both a 327 amino acid residue mono-functionalenzyme and as part of a multi-functional enzyme, also known asbifunctional enzyme, which possesses enoyl-CoA hydratase, enoyl-CoAisomerase, and 3-hydroxyacyl-CoA hydrogenase activities. (FitzPatrick,D. R. et al. (1995) Genomics 27:457-466; and Hoefler, G. et al. (1994)Genomics 19:60-67.) A 339 amino acid residue human protein withshort-chain enoyl-CoA hydratase activity also acts as an AU-specific RNAbinding protein. (Nakagawa, J. et al. (1995) Proc. Natl. Acad. Sci. USA92:2051-2055.) All enoyl-CoA hydratases share homology near two activesite glutamic acid residues, with 17 amino acid residues highlyconserved. (Wu, W.-J. et al. (1997) Biochemistry 36:2211-2220.)

Inherited deficiencies in mitochondrial and peroxisomal beta-oxidationenzymes are associated with severe diseases, some of which manifestthemselves soon after birth and lead to death within a few years.Mitochondrial beta-oxidation associated deficiencies include, e.g.,carnitine palmitoyl transferase and carnitine deficiency,very-long-chain acyl-CoA dehydrogenase deficiency, medium-chain acyl-CoAdehydrogenase deficiency, short-chain acyl-CoA dehydrogenase deficiency,electron transport flavoprotein and electron transportflavoprotein:ubiquinone oxidoreductase deficiency, trifunctional proteindeficiency, and short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency.(Eaton, supra.) Mitochondrial trifunctional protein (including enoyl-CoAhydratase) deficient patients have reduced long-chain enoyl-CoAhydratase activities and suffer from non-ketotic hypoglycemia, suddeninfant death syndrome, cardiomyopathy, hepatic dysfunction, and muscleweakness, and may die at an early age. (Eaton, supra.) A patient with adeficiency in mitochondrial 2,4-dienoyl-CoA reductase was hypotonic soonafter birth, had feeding difficulties, and died at four months fromrespiratory acidosis. (Roe, C. R. et al. (1990) J. Clin. Invest.85:1703-1707.)

Defects in mitochondrial beta-oxidation are associated with Reye'ssyndrome, a disease characterized by hepatic dysfunction andencephalopathy that sometimes follows viral infection in children.Reye's syndrome patients may have elevated serum levels of free fattyacids. (Cotran, R. S. et al. (1994) Robbins Pathologic Basis of Disease,W. B. Saunders Co., Philadelphia, Pa., p.866.) Patients withmitochondrial short-chain 3-hydroxyacyl-CoA dehydrogenase deficiency andmedium-chain 3-hydroxyacyl-CoA dehydrogenase deficiency also exhibitReye-like illnesses. (Eaton, supra; and Egidio, R. J. et al. (1989) Am.Fam. Physician 39:221-226.)

Inherited conditions associated with peroxisomal beta-oxidation includeZellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum'sdisease, acyl-CoA oxidase deficiency, peroxisomal thiolase deficiency,and bifunctional protein deficiency. (Suzuki, Y. et al. (1994) Am. J.Hum. Genet. 54:36-43; Hoefler, supra) Patients with peroxisomalbifunctional enzyme, including enoyl-CoA hydratase, deficiency sufferfrom hypotonia, seizures, psychomotor defects, and defective neuronalmigration; accumulate very-long-chain fatty acids; and typically diewithin a few years of birth. (Watkins, P. A. et al. (1989) J. Clin.Invest. 83:771-777.)

Peroxisomal beta-oxidation is impaired in cancerous tissue. Althoughneoplastic human breast epithelial cells have the same number ofperoxisomes as do normal cells, fatty acyl-CoA oxidase activity is lowerthan in control tissue. (el Bouhtoury, F., et al. (1992) J. Pathol.166:27-35.) Human colon carcinomas have fewer peroxisomes than normalcolon tissue and have lower fatty-acyl-CoA oxidase and bifunctionalenzyme (including enoyl-CoA hydratase) activities than normal tissue.(Cable, S., et al. (1992) Virchows Arch. B Cell Pathol. Incl. Mol.Pathol. 62:221-226.)

The discovery of new human fatty acid beta-oxidation enzymes and thepolynucleotides encoding them satisfies a need in the art by providingnew compositions which are useful in the diagnosis, treatment, andprevention of genetic disorders, neuronal disorders, cancer, infectiousdiseases, liver disorders, and cardiac and skeletal muscle disorders.

SUMMARY OF THE INVENTION

The invention features substantially purified polypeptides, human fattyacid beta-oxidation enzymes, referred to collectively as "HUFA" andindividually as "HUFA-1 " and "HUFA-2." In one aspect, the inventionprovides a substantially purified polypeptide, HUFA, comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO:3, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO:3.

The invention further provides a substantially purified variant of HUFAhaving at least 90% amino acid identity to the amino acid sequences ofSEQ ID NO: 1 or SEQ ID NO:3, or to a fragment of either of thesesequences. The invention also provides an isolated and purifiedpolynucleotide sequence encoding the polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO:3, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO:3. Theinvention also includes an isolated and purified polynucleotide varianthaving at least 90% polynucleotide identity to the polynucleotidesequence encoding the polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, afragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3.

Additionally, the invention provides a composition comprising apolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence selected from the group consisting of SEQ ID NO:1, SEQ IDNO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ ID NO:3. Theinvention further provides an isolated and purified polynucleotidesequence which hybridizes under stringent conditions to thepolynucleotide sequence encoding the polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 1, SEQ IDNO:3, a fragment of SEQ ID NO: 1, and a fragment of SEQ ID NO:3, as wellas an isolated and purified polynucleotide sequence which iscomplementary to the polynucleotide sequence encoding the polypeptidecomprising the amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment ofSEQ ID NO:3.

The invention also provides an isolated and purified polynucleotidesequence comprising a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and afragment of SEQ ID NO:4. The invention further provides an isolated andpurified polynucleotide variant having at least 90% polynucleotideidentity to the polynucleotide sequence comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4,a fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4, as well as anisolated and purified polynucleotide sequence which is complementary tothe polynucleotide sequence comprising a polynucleotide sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, afragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide sequence encoding the polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO:3, a fragment of SEQ ID NO: 1, and a fragment ofSEQ ID NO:3. In another aspect, the expression vector is containedwithin a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, afragment of SEQ D) NO:1, or a fragment of SEQ ID NO:3, the methodcomprising the steps of: (a) culturing the host cell containing anexpression vector containing at least a fragment of a polynucleotidesequence encoding HUFA under conditions suitable for the expression ofthe polypeptide; and (b) recovering the polypeptide from the host cellculture.

The invention also provides a pharmaceutical composition comprising asubstantially purified HUFA having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO: 1, or a fragment of SEQ ID NO:3in conjunction with a suitable pharmaceutical carrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ IDNO:3, a fragment of SEQ ID NO: 1, or a fragment of SEQ ID NO:3, as wellas a purified agonist and a purified antagonist to the polypeptide.

The invention also provides a method for treating or preventing agenetic disorder, the method comprising administering to a subject inneed of such treatment an effective amount of a pharmaceuticalcomposition comprising substantially purified HUFA.

The invention also provides a method for treating or preventing aneuronal disorder, the method comprising administering to a subject inneed of such treatment an effective amount of a pharmaceuticalcomposition comprising substantially purified HUFA-1.

The invention also provides a method for treating or preventing acancer, the method comprising administering to a subject in need of suchtreatment an effective amount of a pharmaceutical composition comprisingsubstantially purified HUFA- 1.

The invention also provides a method for treating or preventing aninfectious disease, the method comprising administering to a subject inneed of such treatment an effective amount of a pharmaceuticalcomposition comprising substantially purified HUFA-2.

The invention also provides a method for treating or preventing a liverdisorder, the method comprising administering to a subject in need ofsuch treatment an effective amount of a pharmaceutical compositioncomprising substantially purified HUFA-2.

The invention also provides a method for treating or preventing acardiac or skeletal muscle disorder, the method comprising administeringto a subject in need of such treatment an effective amount of apharmaceutical composition comprising substantially purified HUFA-2.

The invention also provides a method for detecting a polynucleotideencoding HUFA in a biological sample containing nucleic acids, themethod comprising the steps of: (a) hybridizing the complement of thepolynucleotide sequence encoding the polypeptide comprising SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO: 1, or a fragment of SEQ ID NO:3to at least one of the nucleic acids of the biological sample, therebyforming a hybridization complex; and (b) detecting the hybridizationcomplex, wherein the presence of the hybridization complex correlateswith the presence of a polynucleotide encoding HUFA in the biologicalsample. In one aspect, the nucleic acids of the biological sample areamplified by the polymerase chain reaction prior to the hybridizingstep.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, and 1D show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of HUFA-1. The alignment wasproduced using MACDNASIS PRO™ software (Hitachi Software Engineering Co.Ltd., San Bruno, Calif.).

FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H show the amino acid sequence(SEQ ID NO:3) and nucleic acid sequence (SEQ ID NO:4) of HUFA-2. Thealignment was produced using MACDNASIS PRO™ software.

FIGS. 3A, 3B, and 3C show the amino acid sequence alignments amongHUFA-1 (Incyte Clone 1995961; SEQ ID NO: 1), Saccharomyces cerevisiaeperoxisomal 2,4-dienoyl-CoA reductase (GI 730864; SEQ ID NO:5), humanmitochondrial 2,4-dienoyl-CoA reductase (GI 602703; SEQ ID NO:6), andrat 2,4-dienoyl-CoA reductase (GI 111287; SEQ ID NO:7), produced usingthe multisequence alignment program of LASERGENE software (DNASTAR Inc.,Madison, Wis.).

FIGS. 4A, 4B, and 4C show the amino acid sequence alignments amongHUFA-2 (Incyte Clone 2595635; SEQ ID NO:3), human AU-bindingprotein/enoyl-CoA hydratase (GI 780241; SEQ ID NO:8), human enoyl-CoAhydratase (GI 1922287; SEQ ID NO:9), and human peroxisomal enoyl-CoAhydratase-like protein (GI 564065; SEQ ID NO: 10), produced using themultisequence alignment program of LASEGENE software.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a," "an," and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to "ahost cell" includes a plurality of such host cells, and a reference to"an antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are cited for the purpose of describing and disclosing the celllines, vectors, and methodologies which are reported in the publicationsand which might be used in connection with the invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

DEFINITIONS

"HUFA," as used herein, refers to the amino acid sequences ofsubstantially purified HUFA obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

The term "agonist," as used herein, refers to a molecule which, whenbound to HUFA, increases or prolongs the duration of the effect of HUFA.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to and modulate the effect of HUFA.

An "allele" or an "allelic sequence," as these terms are used herein, isan alternative form of the gene encoding HUFA. Alleles may result fromat least one mutation in the nucleic acid sequence and may result inaltered mRNAs or in polypeptides whose structure or function may or maynot be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toalleles are generally ascribed to natural deletions, additions, orsubstitutions of nucleotides. Each of these types of changes may occuralone, or in combination with the others, one or more times in a givensequence.

"Altered" nucleic acid sequences encoding HUFA, as described herein,include those sequences with deletions, insertions, or substitutions ofdifferent nucleotides, resulting in a polynucleotide the same HUFA or apolypeptide with at least one functional characteristic of HUFA.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding HUFA, and improper or unexpected hybridizationto alleles, with a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding HUFA. The encoded protein may also be"altered," and may contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent HUFA. Deliberate amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of HUFA isretained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, positively charged amino acids mayinclude lysine and arginine, and amino acids with uncharged polar headgroups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; and phenylalanine and tyrosine.

The terms "amino acid" or "amino acid sequence," as used herein, referto an oligopeptide, peptide, polypeptide, or protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. In this context, "fragments", "immunogenic fragments", or"antigenic fragments" refer to fragments of HUFA which are preferablyabout 5 to about 15 amino acids in length and which retain somebiological activity or immunological activity of HUFA. Where "amino acidsequence" is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, "amino acid sequence" and liketerms are not meant to limit the amino acid sequence to the completenative amino acid sequence associated with the recited protein molecule.

"Amplification," as used herein, relates to the production of additionalcopies of a nucleic acid sequence. Amplification is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler (1995) PCRPrimer, a Laboratory Manual, pp. 1-5, Cold Spring Harbor Press,Plainview, N.Y.)

The term "antagonist," as it is used herein, refers to a molecule which,when bound to HUFA, decreases the amount or the duration of the effectof the biological or immunological activity of HUFA. Antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies, or any othermolecules which decrease the effect of HUFA.

As used herein, the term "antibody" refers to intact molecules as wellas to fragments thereof, such as Fab, F(ab')₂, and Fv fragments, whichare capable of binding the epitopic determinant. Antibodies that bindHUFA polypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term "antigenic determinant," as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

The term "antisense," as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to a specificnucleic acid sequence. The term "antisense strand" is used in referenceto a nucleic acid strand that is complementary to the "sense" strand.Antisense molecules may be produced by any method including synthesis ortranscription. Once introduced into a cell, the complementarynucleotides combine with natural sequences produced by the cell to formduplexes and to block either transcription or translation. Thedesignation "negative" can refer to the antisense strand, and thedesignation "positive" can refer to the sense strand.

As used herein, the term "biologically active," refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic HUFA, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms "complementary" or "complementarity," as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base pairing. For example, the sequence"A-G-T" binds to the complementary sequence "T-C-A." Complementaritybetween two single-stranded molecules may be "partial," such that onlysome of the nucleic acids bind, or it may be "complete," such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of the hybridization between the nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands, andin the design and use of peptide nucleic acid (PNA) molecules.

A "composition comprising a given polynucleotide sequence" or a"composition comprising a given amino acid sequence," as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation, an aqueous solution, or a sterile composition.Compositions comprising polynucleotide sequences encoding HUFA orfragments of HUFA may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS), and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

The phrase "consensus sequence," as used herein, refers to a nucleicacid sequence which has been resequenced to resolve uncalled bases,extended using XL-PCR (Perkin Elmer, Norwalk, Conn.) in the 5' and/orthe 3' direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW fragment assemblysystem (GCG, Madison, Wis.). Some sequences have been both extended andassembled to produce the consensus sequence.

As used herein, the term "correlates with expression of apolynucleotide" indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding HUFA, bynorthern analysis is indicative of the presence of nucleic acidsencoding HUFA in a sample, and thereby correlates with expression of thetranscript from the polynucleotide encoding HUFA.

A "deletion," as the term is used herein, refers to a change in theamino acid or nucleotide sequence that results in the absence of one ormore amino acid residues or nucleotides.

The term "derivative," as used herein, refers to the chemicalmodification of HUFA, of a polynucleotide sequence encoding HUFA, or ofa polynucleotide sequence complementary to a polynucleotide sequenceencoding HUFA. Chemical modifications of a polynucleotide sequence caninclude, for example, replacement of hydrogen by an alkyl, acyl, oramino group. A derivative polynucleotide encodes a polypeptide whichretains at least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation, or any similar process that retains a at least onebiological or immunological function of the polypeptide from which itwas derived.

The term "homology," as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology. Theword "identity" may substitute for the word "homology." A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to as"substantially homologous." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially homologous sequence or hybridization probe will competefor and inhibit the binding of a completely homologous sequence to thetarget sequence under conditions of reduced stringency. This is not tosay that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% homology oridentity). In the absence of non-specific binding, the substantiallyhomologous sequence or probe will not hybridize to the secondnon-complementary target sequence.

The phrases "percent identity" or "% identity" refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (LASERGENE softwarepackage, DNASTAR, Inc., Madison Wis.). The MEGALIGN program can createalignments between two or more sequences according to different methods,e.g., the Clustal Method. (Higgins, D. G. and P. M. Sharp (1988) Gene73:237-244.) The Clustal algorithm groups sequences into clusters byexamining the distances between all pairs. The clusters are alignedpairwise and then in groups. The percentage similarity between two aminoacid sequences, e.g., sequence A and sequence B, is calculated bydividing the length of sequence A, minus the number of gap residues insequence A, minus the number of gap residues in sequence B, into the sumof the residue matches between sequence A and sequence B, times onehundred. Gaps of low or of no homology between the two amino acidsequences are not included in determining percentage similarity. Percentidentity between nucleic acid sequences can also be calculated by theClustal Method, or by other methods known in the art, such as the JotunHein Method. (See, e.g., Hein, J. (1990) Methods in Enzymology183:626-645.) Identity between sequences can also be determined by othermethods known in the art, e.g., by varying hybridization conditions.

"Human artificial chromosomes" (HACs), as described herein, are linearmicrochromosomes which may contain DNA sequences of about 6 kb to 10 Mbin size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (Harrington, J. J. etal. (1997) Nat Genet. 15:345-355.)

The term "humanized antibody," as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

"Hybridization," as the term is used herein, refers to any process bywhich a strand of nucleic acid binds with a complementary strand throughbase pairing.

As used herein, the term "hybridization complex" as used herein, refersto a complex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary bases. A hybridizationcomplex may be formed in solution (e.g., C₀ t or R₀ t analysis) orformed between one nucleic acid sequence present in solution and anothernucleic acid sequence immobilized on a solid support (e.g., paper,membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

The words "insertion" or "addition," as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule.

The term "microarray," as used herein, refers to an array of distinctpolynucleotides or oligonucleotides arrayed on a substrate, such aspaper, nylon or any other type of membrane, filter, chip, glass slide,or any other suitable solid support.

The term "modulate," as it appears herein, refers to a change in theactivity of HUFA. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of HUFA.

The phrases "nucleic acid" or "nucleic acid sequence," as used herein,refer to an oligonucleotide, nucleotide, polynucleotide, or any fragmentthereof, to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material. In this context, "fragments" refers to those nucleicacid sequences which are greater than about 60 nucleotides in length,and most preferably are at least about 100 nucleotides, at least about1000 nucleotides, or at least about 10,000 nucleotides in length.

The term "oligonucleotide," as used herein, refers to a nucleic acidsequence of at least about 6 nucleotides to 60 nucleotides, preferablyabout 15 to 30 nucleotides, and most preferably about 20 to 25nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term"oligonucleotide" is substantially equivalent to the terms"amplimers,""primers," "oligomers," and "probes," as these terms arecommonly defined in the art.

"Peptide nucleic acid" (PNA), as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast about 5 nucleotides in length linked to a peptide backbone ofamino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA and RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (Nielsen, P. E. et al.(1993) Anticancer Drug Des. 8:53-63.)

The term "sample," as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acids encoding HUFA,or fragments thereof, or HUFA itself may comprise a bodily fluid; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA (in solution or bound to a solidsupport); a tissue; a tissue print; and the like.

As used herein, the terms "specific binding" or "specifically binding"refer to that interaction between a protein or peptide and an agonist,an antibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein recognized by thebinding molecule (i.e., the antigenic determinant or epitope). Forexample, if an antibody is specific for epitope "A," the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

As used herein, the term "stringent conditions" refers to conditionswhich permit hybridization between polynucleotide sequences and theclaimed polynucleotide sequences. Suitably stringent conditions can bedefined by, for example, the concentrations of salt or formamide in theprehybridization and hybridization solutions, or by the hybridizationtemperature, and are well known in the art. In particular, stringencycan be increased by reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5× SSPE, 0.3%SDS, and 200 μg/ml sheared and denatured salmon sperm DNA. Hybridizationcould occur under reduced stringency conditions as described above, butin 35% formamide at a reduced temperature of 35° C. The temperaturerange corresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

The term "substantially purified," as used herein, refers to nucleicacid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

A "substitution," as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

"Transformation," as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term "transformed" cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, and refers to cells which transiently express the insertedDNA or RNA for limited periods of time.

A "variant" of HUFA, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have "nonconservative" changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE software.

THE INVENTION

The invention is based on the discovery of new human fatty acidbeta-oxidation enzymes (HUFA), the polynucleotides encoding HUFA, andthe use of these compositions for the diagnosis, treatment, orprevention of genetic disorders, neuronal disorders, cancer, infectiousdiseases, liver disorders, and cardiac and skeletal muscle disorders.

Nucleic acids encoding the HUFA- 1 of the present invention were firstidentified in Incyte Clone 1995961 from the human breast tumor cDNAlibrary (BRSTTUT03) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:2, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 270674 (HNT2NOT01), 1995961 (BRSTTUT03), and 2851728 (BRSTTUT13).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1 as shown in FIGS. 1A-1D. HUFA-1is 303 amino acids in length and has a potential peroxisomal C-terminaltargeting signal at A301KL. HUFA-1 has potential casein kinase IIphosphorylation sites at residues S57, S93, S 114, T208, S219, and T296and potential protein kinase C phosphorylation sites at residues S49, S120, and T296. HUFA-1 has a potential NADPH-binding site motif atA21-E53, containing five of the eight consensus amino acid residues:A21, V23, G25, 134, and E53. As shown in FIGS. 3A-3C, HUFA-1 haschemical and structural homology with Saccharomyces cerevisiaeperoxisomal 2,4-dienoyl-CoA reductase (GI 730864; SEQ ID NO:5), humanmitochondrial 2,4-dienoyl-CoA reductase (GI 602703; SEQ ID NO:6), andrat 2,4-dienoyl-CoA reductase (GI 111287; SEQ ID NO:7). In particular,HUFA- 1 and Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoAreductase share 31% identity, HUFA- 1 and human mitochondrial2,4-dienoyl-CoA reductase share 31% identity, and HUFA-1 and rat2,4-dienoyl-CoA reductase share 30% identity. In the region of thepotential NADPH-binding site motif, A21-E53, HUFA-1 is 39% identical toSaccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA reductase, 52%identical to human mitochondrial 2,4-dienoyl-CoA reductase, and 52%identical to rat 2,4-dienoyl-CoA reductase. Northern analysis shows theexpression of this sequence in various libraries, at least 81% of whichare immortalized or cancerous and at least 9% of which involve immuneresponse. Of particular note is the expression of HUFA- 1 in librariesfrom breast and brain tissue.

Nucleic acids encoding the HUFA-2 of the present invention were firstidentified in Incyte Clone 2595635 from the human ovarian tumor cDNAlibrary (OVARTUT02) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:4, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 077607 (SYNORAB01), 416055 (BRSTNOT01), 782840 (MYOMNOT01),1421780 (KIDNNOT09), 1449639 (PLACNOT02), 1474617 (LUNGTUT03), 1485974(CORPNOT02), 1617081 (BRAITUT12), 1987379 (LUNGAST01), and 2595635(OVARTUT02).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:3 as shown in FIGS. 2A-2H. HUFA-2is 301 amino acids in length and has a potential mitochondrialN-terminal leader peptide from residues M1 through Y33. HUFA-2 haspotential casein kinase II phosphorylation sites at residues S107, T119,T161, S229, S235, and S263, and potential protein kinase Cphosphorylation sites at residues S12, T190, and S263. As shown in FIGS.4A-4C, HUFA-2 has chemical and structural homology with human AU-bindingprotein/enoyl-CoA hydratase (GI 780241; SEQ ID NO:8), human enoyl-CoAhydratase (GI 1922287; SEQ ID NO:9), and human peroxisomal enoyl-CoAhydratase-like protein (GI 564065; SEQ ID NO: 10). In particular, HUFA-2and human AU-binding protein/enoyl-CoA hydratase share 25% identity,HUFA-2 and human enoyl-CoA hydratase share 20% identity, and HUFA-2 andhuman peroxisomal enoyl-CoA hydratase-like protein share 21% identity.In particular, 10 of the 17 highly conserved residues around theenoyl-CoA hydratase active site are found in HUFA-2: G150, G154, G155,G156, E158, D164, G182, P185, G189, and G198. In particular, HUFA-2contains the enoyl-CoA hydratase active site residue E158. Northernanalysis shows the expression of this sequence in various libraries, atleast 46% of which are immortalized or cancerous and at least 26% ofwhich involve immune response. Of particular note is the expression ofHUFA-2 in libraries prepared from heart and liver tissue.

The invention also encompasses HUFA variants. A preferred HUFA variantis one which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe HUFA amino acid sequence, and which contains at least one functionalor structural characteristic of HUFA.

The invention also encompasses polynucleotides which encode HUFA. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:2, as shown in FIG. 1,which encodes a HUFA- 1. In a further embodiment, the inventionencompasses the polynucleotide sequence comprising the sequence of SEQID NO:4, as shown in FIG. 2, which encodes a HUFA-2. The invention alsoencompasses a variant of a polynucleotide sequence encoding HUFA. Inparticular, such a variant polynucleotide sequence will have at leastabout 80%, more preferably at least about 90%, and most preferably atleast about 95% polynucleotide sequence identity to the polynucleotidesequence encoding HUFA. A particular aspect of the invention encompassesa variant of SEQ ID NO:2 which has at least about 80%, more preferablyat least about 90%, and most preferably at least about 95%polynucleotide sequence identity to SEQ ID NO:2. The invention furtherencompasses a polynucleotide variant of SEQ ID NO:4 having at leastabout 80%, more preferably at least about 90%, and most preferably atleast about 95% polynucleotide sequence identity to SEQ ID NO:4. Any oneof the polynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of HUFA.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding HUFA, some bearing minimal homology to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring HUFA, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode HUFA and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HUFA under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HUFA or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding HUFA and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences which encodeHUFA and HUFA derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding HUFA or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:2, SEQ ID NO:4, a fragment ofSEQ ID NO:2, or a fragment of SEQ ID NO:4 under various conditions ofstringency as taught in Wahl, G. M. and S. L. Berger (1987; MethodsEnzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.152:507-511).

Methods for DNA sequencing are well known and generally available in theart and may be used to practice any of the embodiments of the invention.The methods may employ such enzymes as the Klenow fragment of DNApolymerase I, SEQUENASE (US Biochemical Corp., Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification systemmarketed by GIBCO/BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the MICROLAB 2200 (Hamilton, Reno,Nev.), Peltier thermal cycler (PTC200; MJ Research, Watertown, Mass.)and the ABI CATALYST and 373 and 377 DNA SEQUENCERS (Perkin Elmer).

The nucleic acid sequences encoding HUFA may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences, such as promoters and regulatoryelements. For example, one method which may be employed,restriction-site PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus. (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322.) In particular, genomic DNA is first amplified in thepresence of a primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region. (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186.) The primers may be designed usingcommercially available software such as OLIGO 4.06 primer analysissoftware (National Biosciences Inc., Plymouth, Minn.) or anotherappropriate program to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to 72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR, which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA. (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119.) In this method, multiple restriction enzymedigestions and ligations may be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR. Another method which may be used to retrieveunknown sequences is that of Parker, J. D. et al. (1991; Nucleic AcidsRes. 19:3055-3060). Additionally, one may use PCR, nested primers, andPROMOTERFINDER libraries to walk genomic DNA (Clontech, Palo Alto,Calif.). This process avoids the need to screen libraries and is usefulin finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will include moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5' non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and a charge coupled device camera for detection of the emittedwavelengths. Output/light intensity may be converted to electricalsignal using appropriate software (e.g., GENETYPER and SEQUENCENAVIGATOR, Perkin Elmer), and the entire process from loading of samplesto computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode HUFA may be used in recombinant DNAmolecules to direct expression of HUFA, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressHUFA.

As will be understood by those of skill in the art, it may beadvantageous to produce HUFA-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter HUFA encodingsequences for a variety of reasons including, but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HUFA may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of HUFA activity, it may be useful toencode a chimeric HUFA protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the HUFA encoding sequence and theheterologous protein sequence, so that HUFA may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding HUFA may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.225-232.) Alternatively, the protein itself may be produced usingchemical methods to synthesize the amino acid sequence of HUFA, or afragment thereof. For example, peptide synthesis can be performed usingvarious solid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved using the ABI 431Apeptide synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography. (See, e.g., Chiez,R. M. and Regnier, F. Z. (1990) Methods Enzymol. 182:392-421.) Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or by sequencing. (See, e.g., the Edman degradation proceduredescribed in Creighton, T. (1983) Proteins, Structures and MolecularPrinciples, WH Freeman and Co., New York, N.Y.). Additionally, the aminoacid sequence of HUFA, or any part thereof, may be altered during directsynthesis and/or combined with sequences from other proteins, or anypart thereof, to produce a variant polypeptide.

In order to express a biologically active HUFA, the nucleotide sequencesencoding HUFA or derivatives thereof may be inserted into appropriateexpression vector, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted coding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding HUFA andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989; Molecular Cloning, A LaboratoryManual, ch. 4, 8, and 16-17, Cold Spring Harbor Press, Plainview, N.Y.)and Ausubel, F. M. et al. (1995 and periodic supplements; CurrentProtocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons,New York, N.Y.).

A variety of expression vector/host systems may be utilized to containand express sequences encoding HUFA. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector (i.e., enhancers, promoters, and 5'and 3' untranslated regions) which interact with host cellular proteinsto carry out transcription and translation. Such elements may vary intheir strength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 (GIBCO/BRL), and the like, may be used. Thebaculovirus polyhedrin promoter may be used in insect cells. Promotersor enhancers derived from the genomes of plant cells (e.g., heat shock,RUBISCO, and storage protein genes) or from plant viruses (e.g., viralpromoters or leader sequences) may be cloned into the vector. Inmammalian cell systems, promoters from mammalian genes or from mammalianviruses are preferable. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding HUFA, vectors based onSV40 or EBV may be used with an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for HUFA. For example, when largequantities of HUFA are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to,multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding HUFA may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced, pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509), and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters, such as alpha factor, alcoholoxidase, and PGH, may be used. For reviews, see Ausubel (supra) andGrant et al. (1987; Methods Enzymol. 153:516-544).

In cases where plant expression vectors are used, the expression ofsequences encoding HUFA may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.) Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used. (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105.) These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews. (See, for example, Hobbs, S. or Murry, L. E. inMcGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, NewYork, N.Y.; pp. 191-196.)

An insect system may also be used to express HUFA. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding HUFA may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of HUFA will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which HUFA may be expressed. (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227.)

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding HUFA may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing HUFA in infected host cells. (Logan, J. and T.Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding HUFA. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding HUFA and its initiation codon and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate for the particularcell system used, such as those described in the literature. (Scharf, D.et al. (1994) Results Probl. Cell Differ. 20:125-162.)

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding,and/or function. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available fromthe American Type Culture Collection (ATCC, Manassas, Va.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

For long term, high yield production of recombinant proteins, stableexpression is preferred. For example, cell lines capable of stablyexpressing HUFA can be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for about 1 to 2 days in enriched media before being switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase genes (Wigler, M. et al. (1977) Cell 11:223-32) andadenine phosphoribosyltransferase genes (Lowy, I. et al. (1980) Cell22:817-23), which can be employed in tk⁻ or apf⁻ cells, respectively.Also, antimetabolite, antibiotic, or herbicide resistance can be used asthe basis for selection. For example, dhfr confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt confers resistance to the aminoglycosides neomycin andG-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and alsor pat confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine. (Hartman, S. C. and R. C.Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51.) Recently, the use ofvisible markers has gained popularity with such markers as anthocyanins,β glucuronidase and its substrate GUS, and luciferase and its substrateluciferin. These markers can be used not only to identify transformants,but also to quantify the amount of transient or stable proteinexpression attributable to a specific vector system. (Rhodes, C.A. etal. (1995) Methods Mol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingHUFA is inserted within a marker gene sequence, transformed cellscontaining sequences encoding HUFA can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding HUFA under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding HUFA and express HUFA may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein sequences.

The presence of polynucleotide sequences encoding HUFA can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding HUFA. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding HUFA to detect transformantscontaining DNA or RNA encoding HUFA.

A variety of protocols for detecting and measuring the expression ofHUFA, using either polyclonal or monoclonal antibodies specific for theprotein, are known in the art. Examples of such techniques includeenzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on HUFA is preferred, but a competitivebinding assay may be employed. These and other assays are well describedin the art, for example, in Hampton, R. et al. (1990; SerologicalMethods, a Laboratory Manual, Section IV, APS Press, St Paul, Minn.) andin Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding HUFA includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding HUFA,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by Pharmacia &Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S. BiochemicalCorp. (Cleveland, Ohio). Suitable reporter molecules or labels which maybe used for ease of detection include radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents, as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding HUFA may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeHUFA may be designed to contain signal sequences which direct secretionof HUFA through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding HUFA to nucleotidesequences encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences, such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.), between the purificationdomain and the HUFA encoding sequence may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing HUFA and a nucleic acid encoding 6 histidineresidues preceding a thioredoxin or an enterokinase cleavage site. Thehistidine residues facilitate purification on immobilized metal ionaffinity chromatography (IMIAC; described in Porath, J. et al. (1992)Prot. Exp. Purif. 3: 263-281)), while the enterokinase cleavage siteprovides a means for purifying HUFA from the fusion protein. Adiscussion of vectors which contain fusion proteins is provided inKroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

Fragments of HUFA may be produced not only by recombinant production,but also by direct peptide synthesis using solid-phase techniques.(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154.) Protein synthesismay be performed by manual techniques or by automation. Automatedsynthesis may be achieved, for example, using the Applied Biosystems431A peptide synthesizer (Perkin Elmer). Various fragments of HUFA maybe synthesized separately and then combined to produce the full lengthmolecule.

THERAPEUTICS

Chemical and structural homology exists among HUFA-1 and peroxisomal2,4-dienoyl-CoA reductase from Saccharomyces cerevisiae (GI 730864),mitochondrial 2,4-dienoyl-CoA reductase from human (GI 602703), and2,4-dienoyl-CoA reductase from rat (GI 111287). In addition, HUFA-1 isexpressed in cancerous and brain tissue. Therefore, HUFA-1 appears toplay a role in genetic disorders, neuronal disorders, and cancer.

Chemical and structural homology exists among HUFA-2 and AU-bindingprotein/enoyl-CoA hydratase from human (GI 780241), enoyl-CoA hydratasefrom human (GI 1922287), and peroxisomal enoyl-CoA hydratase-likeprotein from human (GI 564065). In addition, HUFA-2 is expressed inheart and liver tissue. Therefore, HUFA-2 appears to play a role ingenetic disorders, infectious diseases, liver disorders, and cardiac andskeletal muscle disorders.

Therefore, in one embodiment, HUFA or a fragment or derivative thereofmay be administered to a subject to treat or prevent a genetic disorder.Such disorders can include, but are not limited to,adrenoleukodystrophy, Alport's syndrome, choroideremia, Duchenne andBecker muscular dystrophy, Down's syndrome, cystic fibrosis, chronicgranulomatous disease, Gaucher's disease, Huntington's chorea, Marfan'ssyndrome, muscular dystrophy, myotonic dystrophy, pycnodysostosis,Refsum's syndrome, retinoblastoma, sickle cell anemia, thalassemia,Werner syndrome, von Willebrand's disease, Wilm's tumor, Zellwegersyndrome, peroxisomal acyl-CoA oxidase deficiency, peroxisomal thiolasedeficiency, peroxisomal bifunctional protein deficiency, mitochondrialcarnitine palmitoyl transferase and carnitine deficiency, mtiochondrialvery-long-chain acyl-CoA dehydrogenase deficiency, mitochondrialmedium-chain acyl-CoA dehydrogenase deficiency, mitochondrialshort-chain acyl-CoA dehydrogenase deficiency, mitochondrial electrontransport flavoprotein and electron transport flavoprotein:ubiquinoneoxidoreductase deficiency, mitochondrial trifunctional proteindeficiency, and mitochondrial short-chain 3-hydroxyacyl-CoAdehydrogenase deficiency.

In another embodiment, a vector capable of expressing HUFA or a fragmentor derivative thereof may be administered to a subject to treat orprevent a genetic disorder including, but not limited to, thosedescribed above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HUFA in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a genetic disorder including, but not limited to, those providedabove.

In still another embodiment, an agonist which modulates the activity ofHUFA may be administered to a subject to treat or prevent a geneticdisorder including, but not limited to, those listed above.

In another embodiment, HUFA- 1 or a fragment or derivative thereof maybe administered to a subject to treat or prevent a neuronal disorder.Such disorders can include, but are not limited to, akathesia,Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolardisorder, catatonia, cerebral neoplasms, dementia, depression, diabeticneuropathy, Down's syndrome, tardive dyskinesia, dystonias, epilepsy,Huntington's disease, multiple sclerosis, neurofibromatosis, Parkinson'sdisease, paranoid psychoses, postherpetic neuralgia, schizophrenia, andTourette's disorder.

In another embodiment, a vector capable of expressing HUFA-1 or afragment or derivative thereof may be administered to a subject to treator prevent a neuronal disorder including, but not limited to, thosedescribed above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HUFA-1 in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a neuronal disorder including, but not limited to, thoseprovided above.

In still another embodiment, an agonist which modulates the activity ofHUFA-1 may be administered to a subject to treat or prevent a neuronaldisorder including, but not limited to, those listed above.

In another embodiment, HUFA-1 or a fragment or derivative thereof may beadministered to a subject to treat or prevent a cancer. Cancers caninclude, but are not limited to, adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancersof the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus.

In another embodiment, a vector capable of expressing HUFA-1 or afragment or derivative thereof may be administered to a subject to treator prevent a cancer including, but not limited to, those describedabove.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HUFA-1 in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a cancer including, but not limited to, those provided above.

In still another embodiment, an agonist which modulates the activity ofHUFA may be administered to a subject to treat or prevent a cancerincluding, but not limited to, those listed above.

In another embodiment, HUFA-2 or a fragment or derivative thereof may beadministered to a subject to treat or prevent an infectious disease.Such diseases can include, but are not limited to, viral infections:adenoviruses (ARD, pneumonia), arenaviruses (lymphocyticchoriomeningitis), bunyaviruses (Hantavirus), coronaviruses (pneumonia,chronic bronchitis), hepadnaviruses (hepatitis), herpesviruses (HSV,VZV, Epstein-Barr virus, cytomegalovirus), flaviviruses (yellow fever),orthomyxoviruses (influenza), papillomaviruses (cancer), paramyxoviruses(measles, mumps), picornoviruses (rhinovirus, poliovirus,coxsackie-virus), polyomaviruses (BK virus, JC virus), poxviruses(smallpox), reovirus (Colorado tick fever), retroviruses (HIV, HTLV),rhabdoviruses (rabies), rotaviruses (gastroenteritis), and togaviruses(encephalitis, rubella); bacterial infections, fungal infections,parasitic infections, protozoal infections, and helminthic infections.

In another embodiment, a vector capable of expressing HUFA-2 or afragment or derivative thereof may be administered to a subject to treator prevent an infectious disease including, but not limited to, thosedescribed above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HUFA-2 in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent an infectious disease including, but not limited to, thoseprovided above.

In still another embodiment, an agonist which modulates the activity ofHUFA-2 may be administered to a subject to treat or prevent aninfectious disease including, but not limited to, those listed above.

In another embodiment, HUFA-2 or a fragment or derivative thereof may beadministered to a subject to treat or prevent a liver disorder. Suchdisorders can include, but are not limited to, cirrhosis, jaundice,cholestasis, hereditary hyperbilirubinemia, hepatic encephalopathy,hepatorenal syndrome, hepatitis, hepatic steatosis, hemochromatosis,Wilson's disease, alpha,-antitrypsin deficiency, Reye's syndrome,primary sclerosing cholangitis, liver infarction, portal veinobstruction and thrombosis, passive congestion, centrilobular necrosis,peliosis hepatis, hepatic vein thrombosis, veno-occlusive disease,preeclampsia, eclampsia, acute fatty liver of pregnancy, intrahepaticcholestasis of pregnancy, and hepatic tumors including nodularhyperplasias, adenomas, and carcinomas.

In another embodiment, a vector capable of expressing HUFA-2 or afragment or derivative thereof may be administered to a subject to treator prevent a liver disorder including, but not limited to, thosedescribed above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HUFA-2 in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a liver disorder including, but not limited to, those providedabove.

In still another embodiment, an agonist which modulates the activity ofHUFA-2 may be administered to a subject to treat or prevent a liverdisorder including, but not limited to, those listed above.

In another embodiment, HUFA-2 or a fragment or derivative thereof may beadministered to a subject to treat or prevent a cardiac or skeletalmuscle disorder. Such disorders can include, but are not limited to,cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker'smuscular dystrophy, myotonic dystrophy, central core disease, nemalinemyopathy, centronuclear myopathy, lipid myopathy, mitochondrialmyopathy, infectious myositis, polymyositis, dermatomyositis, inclusionbody myositis, thyrotoxic myopathy, and ethanol myopathy.

In another embodiment, a vector capable of expressing HUFA-2 or afragment or derivative thereof may be administered to a subject to treator prevent a cardiac or skeletal muscle disorder including, but notlimited to, those described above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HUFA-2 in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a cardiac or skeletal muscle disorder including, but not limitedto, those provided above.

In still another embodiment, an agonist which modulates the activity ofHUFA-2 may be administered to a subject to treat or prevent a cardiac orskeletal muscle disorder including, but not limited to, those listedabove.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of HUFA may be produced using methods which are generallyknown in the art. In particular, purified HUFA may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind HUFA. Antibodies to HUFA may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith HUFA or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to HUFA have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of HUFA amino acidsmay be fused with those of another protein, such as KLH, and antibodiesto the chimeric molecule may be produced.

Monoclonal antibodies to HUFA may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al. (1984) Mol. CellBiol. 62:109-120.)

In addition, techniques developed for the production of "chimericantibodies," such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (Morrison, S. L. et al. (1984)Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984)Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.)Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceHUFA-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries.(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-11123.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837, andWinter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for HUFA mayalso be generated. For example, such fragments include, but are notlimited to, F(ab)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab)2 fragments. Alternatively, Fab expression librariesmay be constructed to allow rapid and easy identification of monoclonalFab fragments with the desired specificity. (Huse, W. D. et al. (1989)Science 254:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between HUFA and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HUFA epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

In another embodiment of the invention, the polynucleotides encodingHUFA, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingHUFA may be used in situations in which it would be desirable to blockthe transcription of the MRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding HUFA. Thus,complementary molecules or fragments may be used to modulate HUFAactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments can be designed from various locations along the codingor control regions of sequences encoding HUFA.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencecomplementary to the polynucleotides of the gene encoding HUFA. Thesetechniques are described, for example, in Sambrook (supra) and inAusubel (supra).

Genes encoding HUFA can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof encoding HUFA. Such constructs may be used to introduceuntranslatable sense or antisense sequences into a cell. Even in theabsence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5', or regulatory regions of the gene encodingHUFA. Oligonucleotides derived from the transcription initiation site,e.g., between about positions -10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (Gee, J. E. et al. (1994) in Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, pp. 163-177,Futura Publishing Co., Mt. Kisco, N.Y.) A complementary sequence orantisense molecule may also be designed to block translation of MRNA bypreventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules specifically andefficiently catalyze endonucleolytic cleavage of sequences encodingHUFA.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding HUFA. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA constitutivelyor inducibly can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art, such as those described in Goldman, C. K. et al.(1997; Nature Biotechnology 15:462-466).

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of HUFA,antibodies to HUFA, and mimetics, agonists, antagonists, or inhibitorsof HUFA. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acids. Saltstend to be more soluble in aqueous or other protonic solvents than arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of HUFA, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays of neoplastic cells, forexample, or in animal models, usually mice, rabbits, dogs, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example HUFA or fragments thereof, antibodies of HUFA,and agonists, antagonists or inhibitors of HUFA, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED50 (the dosetherapeutically effective in 50% of the population) or LD50 (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe LD50/ED50 ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies is used in formulating a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on the half-life and clearancerate of the particular formulation.

Normal dosage amounts may vary from 0.1 μg to 100,000 μg, up to a totaldose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind HUFA may beused for the diagnosis of disorders characterized by expression of HUFA,or in assays to monitor patients being treated with HUFA or agonists,antagonists, and inhibitors of HUFA. Antibodies useful for diagnosticpurposes may be prepared in the same manner as those described above fortherapeutics. Diagnostic assays for HUFA include methods which utilizethe antibody and a label to detect HUFA in human body fluids or inextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent joiningwith a reporter molecule. A wide variety of reporter molecules, severalof which are described above, are known in the art and may be used.

A variety of protocols for measuring HUFA, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of HUFA expression. Normal or standard values for HUFAexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toHUFA under conditions suitable for complex formation. The amount ofstandard complex formation may be quantified by various methods,preferably by photometric means. Quantities of HUFA expressed insubject, samples from biopsied tissues are compared with the standardvalues. Deviation between standard and subject values establishes theparameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingHUFA may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHUFA may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of HUFA,and to monitor regulation of HUFA levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HUFA or closely related molecules may be used to identifynucleic acid sequences which encode HUFA. The specificity of the probe,whether it is made from a highly specific region (e.g., the 5'regulatory region) or from a less specific region (e.g., the 3' codingregion), and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding HUFA, alleles, orrelated sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe HUFA encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequences of SEQID NO:2 and SEQ ID NO:4 or from genomic sequences including promoter andenhancer elements and introns of the naturally occurring HUFA.

Means for producing specific hybridization probes for DNAs encoding HUFAinclude the cloning of polynucleotide sequences encoding HUFA or HUFAderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³² P or ³⁵ S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding HUFA may be used for the diagnosis ofa disorder associated with expression of HUFA. Examples of suchdisorders include, but are not limited to, genetic disorders, such asadrenoleukodystrophy, Alport's syndrome, choroideremia, Duchenne andBecker muscular dystrophy, Down's syndrome, cystic fibrosis, chronicgranulomatous disease, Gaucher's disease, Huntington's chorea, Marfan'ssyndrome, muscular dystrophy, myotonic dystrophy, pycnodysostosis,Refsum's syndrome, retinoblastoma, sickle cell anemia, thalassemia,Werner syndrome, von Willebrand's disease, Wilm's tumor, Zellwegersyndrome, peroxisomal acyl-CoA oxidase deficiency, peroxisomal thiolasedeficiency, peroxisomal bifunctional protein deficiency, mitochondrialcarnitine palmitoyl transferase and carnitine deficiency, mtiochondrialvery-long-chain acyl-CoA dehydrogenase deficiency, mitochondrialmedium-chain acyl-CoA dehydrogenase deficiency, mitochondrialshort-chain acyl-CoA dehydrogenase deficiency, mitochondrial electrontransport flavoprotein and electron transport flavoprotein:ubiquinoneoxidoreductase deficiency, mitochondrial trifunctional proteindeficiency, and mitochondrial short-chain 3-hydroxyacyl-CoAdehydrogenase deficiency.

Polynucleotide sequences encoding HUFA-1 may be used for the diagnosisof a disorder associated with expression of HUFA-1. Examples of suchdisorders include, but are not limited to, neuronal disorders, such asakathesia, Alzheimer's disease, amnesia, amyotrophic lateral sclerosis,bipolar disorder, catatonia, cerebral neoplasms, dementia, depression,diabetic neuropathy, Down's syndrome, tardive dyskinesia, dystonias,epilepsy, Huntington's disease, multiple sclerosis, neurofibromatosis,Parkinson's disease, paranoid psychoses, postherpetic neuralgia,schizophrenia, and Tourette's disorder; and cancers, includingadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus.

Polynucleotide sequences encoding HUFA-2 may be used for the diagnosisof a disorder associated with expression of HUFA-2. Examples of suchdisorders include, but are not limited to, infectious diseases,including viral infections: adenoviruses (ARD, pneumonia), arenaviruses(lymphocytic choriomeningitis), bunyaviruses (Hantavirus), coronaviruses(pneumonia, chronic bronchitis), hepadnaviruses (hepatitis),herpesviruses (HSV, VZV, Epstein-Barr virus, cytomegalovirus),flaviviruses (yellow fever), orthomyxoviruses (influenza),papillomaviruses (cancer), paramyxoviruses (measles, mumps),picomoviruses (rhinovirus, poliovirus, coxsackie-virus), polyomaviruses(BK virus, JC virus), poxviruses (smallpox), reovirus (Colorado tickfever), retroviruses (HIV, HTLV), rhabdoviruses (rabies), rotaviruses(gastroenteritis), and togaviruses (encephalitis, rubella); bacterialinfections, fungal infections, parasitic infections, protozoalinfections, and helminthic infections; liver disorders, includingcirrhosis, jaundice, cholestasis, hereditary hyperbilirubinemia, hepaticencephalopathy, hepatorenal syndrome, hepatitis, hepatic steatosis,hemochromatosis, Wilson's disease, alpha₁ -antitrypsin deficiency,Reye's syndrome, primary sclerosing cholangitis, liver infarction,portal vein obstruction and thrombosis, passive congestion,centrilobular necrosis, peliosis hepatis, hepatic vein thrombosis,veno-occlusive disease, preeclampsia, eclampsia, acute fatty liver ofpregnancy, intrahepatic cholestasis of pregnancy, and hepatic tumorsincluding nodular hyperplasias, adenomas, and carcinomas; and cardiacand skeletal muscle disorders, including cardiomyopathy, myocarditis,Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonicdystrophy, central core disease, nemaline myopathy, centronuclearmyopathy, lipid myopathy, mitochondrial myopathy, infectious myositis,polymyositis, dermatomyositis, inclusion body myositis, thyrotoxicmyopathy, and ethanol myopathy.

The polynucleotide sequences encoding HUFA may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; in dipstick, pin, and ELISA assays; and in microarraysutilizing fluids or tissues from patient biopsies to detect altered HUFAexpression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences encoding HUFA may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingHUFA may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered from that of a comparable control sample, the nucleotidesequences have hybridized with nucleotide sequences in the sample, andthe presence of altered levels of nucleotide sequences encoding HUFA inthe sample indicates the presence of the associated disorder. Suchassays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies, in clinical trials, orin monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of HUFA, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding HUFA, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for a disorder. Deviation from standard values isused to establish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding HUFA may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding HUFA, or a fragment of a polynucleotide complementary to thepolynucleotide encoding HUFA, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of HUFAinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244, and Duplaa,C. et al. (1993) Anal. Biochem. 212: 229-236.) The speed of quantitationof multiple samples may be accelerated by running the assay in an ELISAformat where the oligomer of interest is presented in various dilutionsand a spectrophotometric or colorimetric response gives rapidquantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously (to produce atranscript image) and to identify genetic variants, mutations, andpolymorphisms. This information may be used in determining genefunction, in understanding the genetic basis of a disorder, indiagnosing a disorder, and in developing and monitoring the activitiesof therapeutic agents.

In one embodiment, the microarray is prepared and used according tomethods known in the art, such as those described in published PCTapplication WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat.Biotech. 14:1675-1680), and Schena, M. et al. (1996; Proc. Natl. Acad.Sci. 93:10614-10619).

The microarray is preferably composed of a large number of uniquesingle-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6 to 60 nucleotidesin length, more preferably about 15 to 30 nucleotides in length, andmost preferably about 20 to 25 nucleotides in length. For a certain typeof microarray, it may be preferable to use oligonucleotides which areabout 7 to 10 nucleotides in length. The microarray may containoligonucleotides which cover the known 5' or 3' sequence, or may containsequential oligonucleotides which cover the full length sequence orunique oligonucleotides selected from particular areas along the lengthof the sequence. Polynucleotides used in the microarray may beoligonucleotides specific to a gene or genes of interest in which atleast a fragment of the sequence is known or oligonucleotides specificto one or more unidentified cDNAs common to a particular cell or tissuetype or to a normal, developmental, or disease state. In certainsituations, it may be appropriate to use pairs of oligonucleotides on amicroarray. The pairs will be identical, except for one nucleotidepreferably located in the center of the sequence. The secondoligonucleotide in the pair (mismatched by one) serves as a control. Thenumber of oligonucleotide pairs may range from about 2 to 1,000,000.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5' end, or, more preferably, at the 3' end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In one aspect, the oligomers aresynthesized at designated areas on a substrate using a light-directedchemical process. The substrate may be paper, nylon, any other type ofmembrane, filter, chip, glass slide, or any other suitable solidsupport.

In one aspect, the oligonucleotides may be synthesized on the surface ofthe substrate by using a chemical coupling procedure and an ink jetapplication apparatus, such as that described in published PCTapplication WO95/251116 (Baldeschweiler et al.). In another aspect, agrid array analogous to a dot or slot blot (HYBRIDOT apparatus,GIBCO/BRL) may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system orthermal, UV, mechanical or chemical bonding procedures. In yet anotheraspect, an array may be produced by hand or by using available devices,materials, and machines (including Brinkmann multichannel pipettors orrobotic instruments), and may contain 8, 24, 96, 384, 1536, or 6144oligonucleotides, or any other multiple from 2 to 1,000,000 which lendsitself to the efficient use of commercially available instrumentation.

In order to conduct sample analysis using the microarrays,polynucleotides are extracted from a biological sample. The biologicalsamples may be obtained from any bodily fluid (blood, urine, saliva,phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissuepreparations. To produce probes, the polynucleotides extracted from thesample are used to produce nucleic acid sequences which arecomplementary to the nucleic acids on the microarray. If the microarrayconsists of cDNAs, antisense RNAs (aRNA) are appropriate probes.Therefore, in one aspect, mRNA is used to produce cDNA which, in turnand in the presence of fluorescent nucleotides, is used to producefragment or oligonucleotide aRNA probes. These fluorescently labeledprobes are incubated with the microarray so that the probe sequenceshybridize to the cDNA oligonucleotides of the microarray. In anotheraspect, nucleic acid sequences used as probes can includepolynucleotides, fragments, and complementary or antisense sequencesproduced using restriction enzymes, PCR technologies, and Oligolabelingor TransProbe kits (Pharmacia & Upjohn) well known in the area ofhybridization technology.

Incubation conditions are adjusted so that hybridization occurs withprecise complementary matches or with various degrees of lesscomplementarity. After removal of nonhybridized probes, a scanner isused to determine the levels and patterns of fluorescence. The scannedimages are examined to determine the degree of complementarity and therelative abundance of each oligonucleotide sequence on the microarray. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies or for functional analysis of the sequences, mutations,variants, or polymorphisms among samples. (Heller, R. A. et al. (1997)Proc. Natl. Acad. Sci. 94:2150-2155.)

In another embodiment of the invention, nucleic acid sequences encodingHUFA may be used to generate hybridization probes useful for mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, such as human artificialchromosomes (HACs), yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), bacterial P1 constructions, or singlechromosome cDNA libraries, such as those reviewed in Price, C. M. (1993;Blood Rev. 7:127-134) and Trask, B. J. (1991; Trends Genet. 7:149-154).

Fluorescent in situ hybridization (FISH, as described, e.g., inHeinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular Biology andBiotechnology, pp. 965-968, VCH Publishers New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in variousscientific journals or at the Online Mendelian Inheritance in Man (OMIM)site. Correlation between the location of the gene encoding HUFA on aphysical chromosomal map and a specific disorder, or predisposition to aspecific disorder, may help define the region of DNA associated withthat disorder. The nucleotide sequences of the subject invention may beused to detect differences in gene sequences between normal, carrier,and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, ataxia-telangiectasia (AT) to 11q22-23 (Gatti, R. A. et al. (1988) Nature 336:577-580), any sequencesmapping to that area may represent associated or regulatory genes forfurther investigation. The nucleotide sequence of the subject inventionmay also be used to detect differences in the chromosomal location dueto translocation, inversion, etc., among normal, carrier, or affectedindividuals.

In another embodiment of the invention, HUFA, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between HUFAand the agent being tested may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564 (Geysen, et al.). In this method, large numbers of differentsmall test compounds are synthesized on a solid substrate, such asplastic pins or some other surface. The test compounds are reacted withHUFA, or fragments thereof, and washed. Bound HUFA is then detected bymethods well known in the art. Purified HUFA can also be coated directlyonto plates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding HUFA specificallycompete with a test compound for binding HUFA. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with HUFA.

In additional embodiments, the nucleotide sequences which encode HUFAmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I. cDNA Library Construction

BRSTTUT03

The BRSTTUT03 cDNA library was constructed from cancerous breast tissueremoved from a 58-year-old Caucasian female who had undergone unilateralextended simple mastectomy following diagnosis of multicentric invasivegrade 4 mammary lobular carcinoma.

The frozen tissue was homogenized and lysed using a Polytron PT-3000homogenizer (Brinkmann Instruments, Westbury, N.J.) in guanidiniumisothiocyanate solution. The lysate was centrifuged over a 5.7 M CsClcushion using an SW28 rotor in an L8-70M ultracentrifuge (BeckmanInstruments) for 18 hours at 25,000 rpm at ambient temperature. The RNAwas extracted with acid phenol pH 4.0, precipitated using 0.3 M sodiumacetate and 2.5 volumes of ethanol, resuspended in RNAse-free water andDNase treated at 37° C. The RNA extraction and precipitation wererepeated as before. The mRRNA was then isolated using the OLIGOTER kit(QIAGEN Inc., Chatsworth, Calif.) and used to construct the cDNAlibrary.

The mRNA was handled according to the recommended protocols in theSUPERSCRIPT plasmid system for cDNA synthesis and plasmid cloning(Catalog #18248-013; Gibco/BRL), cDNAs were fractionated on a SEPHAROSECL4B column (Catalog #275105-01; Pharmacia), and those cDNAs exceeding400 bp were ligated into PSPORT I. The plasmid PSPORT I was subsequentlytransformed into DH5α competent cells (Catalog #18258-012; Gibco/BRL).

OVARTUT02

The OVARTUT02 library was constructed from tumorous ovary tissueobtained from a 51 -year-old Caucasian female during a total abdominalhysterectomy.

The frozen tissue was homogenized and lysed in TRIZOL reagent (1 gtissue/10 ml TRIZOL; Catalog #10296-028; Gibco/BRL), a monoplasticsolution of phenol and guanidine isothiocyanate, using a PolytronPT-3000 homogenizer (Brinkmann Instruments, Westbury, N.Y.). After abrief incubation on ice, chloroform was added (1:5 v/v) and the lysatewas centrifuged. The upper chloroform layer was removed to a fresh tubeand the RNA extracted with isopropanol, resuspended in DEPC-treatedwater, and DNase treated for 25 min at 37° C. The RNA was extracted oncewith acid phenol-chloroform pH 4.7 and precipitated using 0.3M sodiumacetate and 2.5 volumes ethanol. The mRNA was then isolated using theOLIGOTEX kit (QIAGEN) and used to construct the cDNA library.

The mRNA was handled according to the recommended protocols in theSUPERSCRIPT plasmid system for cDNA Synthesis and plasmid cloning(Catalog #18248-013, Gibco/BRL).The cDNAs were fractionated on aSEPHAROSE CL4B column (Catalog #275105-01; Pharmacia), and those cDNAsexceeding 400 bp were ligated into pINCY 1 (Incyte). The plasmid pINCY 1was subsequently transformed into DH5α™ competent cells (Catalog#18258-012; Gibco/BRL).

II. Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the R.E.A.L.PREP 96 plasmid kit (Catalog #26173; QIAGEN). This kit enables thesimultaneous purification of 96 samples in a 96-well block usingmulti-channel reagent dispensers. The recommended protocol was employedexcept for the following changes: 1) the bacteria were cultured in 1 mlof sterile Terrific Broth (Catalog #22711, Gibco/BRL) with carbenicillinat 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

The cDNAs were sequenced by the method of Sanger et al. (1975; J. Mol.Biol. 94:441f), using a MICROLAB 2200 (Hamilton, Reno, Nev.) incombination with Peltier PTC200 thermal cycler (MJ Research, Watertown,Mass.) and Applied Biosystems 377 DNA sequencing systems; and thereading frame was determined.

III. Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences and/or amino acid sequences of the SequenceListing were used to query sequences in the GenBank, SwissProt, BLOCKS,and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofhomology using BLAST (Basic Local Alignment Search Tool). (Altschul, S.F. (1993) J. Mol. Evol 36:290-300; and Altschul et al. (1990) J. Mol.Biol. 215:403-410.)

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms such asthe one described in Smith, T. et al. (1992; Protein Engineering5:35-51), could have been used when dealing with primary sequencepatterns and secondary structure gap penalties. The sequences disclosedin this application have lengths of at least 49 nucleotides and have nomore than 12% uncalled bases (where N is recorded rather than A, C, G,or T).

The BLAST approach searched for matches between a query sequence and adatabase sequence. BLAST evaluated the statistical significance of anymatches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻¹⁰ for peptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and other mammalian sequences (mam),and deduced amino acid sequences from the same clones were then searchedagainst GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp), for homology.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (Sambrook, supra, ch. 7; and Ausubel,supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST are used to search foridentical or related molecules in nucleotide databases such as theGenBank or the LIFESEQ database (Incyte Pharmaceuticals). This analysisis much faster than multiple membrane-based hybridizations. In addition,the sensitivity of the computer search can be modified to determinewhether any particular match is categorized as exact or homologous.

The basis of the search is the product score, which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and, with a product score of 70, the match will beexact. Homologous molecules are usually identified by selecting thosewhich show product scores between 15 and 40, although lower scores mayidentify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding HUFA occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V. Extension of HUFA Encoding Polynucleotides

The nucleic acid sequences of Incyte Clones 1995961 and 2595635 wereused to design oligonucleotide primers for extending partial nucleotidesequences to full length. For each nucleic acid sequence, one primer wassynthesized to initiate extension of an antisense polynucleotide, andthe other was synthesized to initiate extension of a sensepolynucleotide. Primers were used to facilitate the extension of theknown sequence "outward" generating amplicons containing new unknownnucleotide sequence for the region of interest. The initial primers weredesigned from the cDNA using OLIGO 4.06 software (National Biosciences),or another appropriate program, to be about 22 to 30 nucleotides inlength, to have a GC content of about 50% or more, and to anneal to thetarget sequence at temperatures of about 68° C. to about 72° C. Anystretch of nucleotides which would result in hairpin structures andprimer-primer dimerizations was avoided.

Selected human cDNA libraries (GIBCO/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. PCR was performed using the Peltier (PTC200 thermal cyclerM.J. Research, Watertown, Mass.), beginning with 40 pmol of each primerand the recommended concentrations of all other components of the kit,with the following parameters:

    ______________________________________                                        Step 1   94° C. for 1 min (initial denaturation)                                 Step 2 65° C. for 1 min                                        Step 3 68° C. for 6 min                                                Step 4 94° C. for 15 sec                                               Step 5 65° C. for 1 min                                                Step 6 68° C. for 7 min                                                Step 7 Repeat steps 4 through 6 for an additional 15 cycles                   Step 8 94° C. for 15 sec                                               Step 9 65° C. for 1 min                                                Step 10 68° C. for 7:15 min                                            Step 11 Repeat steps 8 through 10 for an additional 12 cycles                 Step 12 72° C. for 8 min                                               Step 13  4° C. (and holding)                                         ______________________________________                                    

A 5 μl to 10μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQUICK (QIAGEN), and trimmed of overhangsusing Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (Sambrook, supra,Appendix A, p. 2.) After incubation for one hour at 37° C., the E. colimixture was plated on Luria Bertani (LB) agar (Sambrook, supra, AppendixA, p. 1) containing 2× Carb. The following day, several colonies wererandomly picked from each plate and cultured in 150 μl of liquid LB/2×Carb medium placed in an individual well of an appropriatecommercially-available sterile 96-well plate. The following day, 5 μl ofeach overnight culture was transferred into a non-sterile 96-well plateand, after dilution 1:10 with water, 5 μl from each sample wastransferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

    ______________________________________                                        Step 1    94° C. for 60 sec                                              Step 2 94° C. for 20 sec                                               Step 3 55° C. for 30 sec                                               Step 4 72° C. for 90 sec                                               Step 5 Repeat steps 2 through 4 for an additional 29 cycles                   Step 6 72° C. for 180 sec                                              Step 7  4° C. (and holding)                                          ______________________________________                                    

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequences of SEQ ID NO:2 and SEQ ID NO:4are used to obtain 5' regulatory sequences using the procedure above,oligonucleotides designed for 5' extension, and an appropriate genomiclibrary.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 and SEQ ID NO:4 areemployed to screen cDNAs, genomic DNAs, or mRNAs. Although the labelingof oligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer and 250 μCi of [λ-³² P] adenosinetriphosphate (Amersham) and T4 polynucleotide kinase (DuPont NEN,Boston, Mass.). The labeled oligonucleotides are substantially purifiedusing a SEPHADEX G-25 superfine resin column (Pharmacia & Upjohn). Analiquot containing 10⁷ counts per minute of the labeled probe is used ina typical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases: Ase I, Bgl II, Eco RI,Pst I, Xba 1, or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (NYTRAN Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film onother film (Kodak, Rochester, N.Y.) is exposed to the blots or the blotsare exposed to a PhosphorImager cassette (Molecular Dynamics, Sunnyvale,Calif.) hybridization patterns are compared visually.

VII. Microarrays

To produce oligonucleotides for a microarray, one of the nucleotidesequences of the present invention is examined using a computeralgorithm which starts at the 3' end of the nucleotide sequence. Thealgorithm identifies oligomers of defined length that are unique to thegene, have a GC content within a range suitable for hybridization, andlack predicted secondary structure that would interfere withhybridization. The algorithm identifies approximately 20sequence-specific oligonucleotides of 20 nucleotides in length(20-mers). A matched set of oligonucleotides are created in which onenucleotide in the center of each sequence is altered. This process isrepeated for each gene in the microarray, and double sets of twenty20-mers are synthesized and arranged on the surface of the silicon chipusing a light-directed chemical process, such as that described in Chee(supra).

In the alternative, a chemical coupling procedure and an ink jet deviceare used to synthesize oligomers on the surface of a substrate. (SeeBaldeschweiler, supra.) In another alternative, a grid array analogousto a dot or slot blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system orthermal, UV, mechanical, or chemical bonding procedures. A typical arraymay be produced by hand or using available materials and machines andcontain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots, or 6144dots. After hybridization, the microarray is washed to removenonhybridized probes, and a scanner is used to determine the levels andpatterns of fluorescence. The scanned image is examined to determine thedegree of complementarity and the relative abundance/expression level ofeach oligonucleotide sequence in the microarray.

VIII. Complementary Polynucleotides

Sequences complementary to the HUFA-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring HUFA. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software andthe coding sequence of HUFA. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5' sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the HUFA-encoding transcript.

IX. Expression of HUFA

Expression of HUFA is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express HUFA in E. coli.This vector contains a promoter for B-galactosidase upstream of thecloning site, followed by sequence containing the amino-terminal Met andthe subsequent seven residues of β-galactosidase. Immediately followingthese eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with isopropylbeta-D-thiogalactopyranoside (IPTG) using standard methods produces afusion protein which consists of the first 8 residues ofβ-galactosidase, about 5 to 15 residues of linker, and the full lengthprotein. The signal residues direct the secretion of HUFA into bacterialgrowth media which can be used directly in the following assay foractivity.

X. Demonstration of HUFA Activity

HUFA-1

HUFA-1 is assayed by measuring the disappearance of5-phenyl-2,4-pentadienoyl-CoA and NADPH, which absorb at 340 nm. (Nada,M. A. et al. (1994) Lipids 29:517-521.) A solution of 0.2 M potassiumphosphate (pH 8), 0.1 mM NADPH, and 25 μM 5-phenyl-2,4,-pentadienoyl-CoAis mixed in an optical cuvette. The assay is started by the addition ofsample to the cuvette. The change in absorbance at 340 nm is measuredusing an ultraviolet spectrophotometer. The amount of HUFA-1 in thesample is proportional to the decrease in absorbance at 340 nm at 25° C.

HUFA-2

HUFA-2 is assayed in a coupled assay measuring the hydration andsubsequent dehydrogenation of either a short-chain enoyl-CoA(crotonyl-CoA) or a long-chain enoyl-CoA (trans-2-dodecenoyl-CoA).(Wanders, R. J. A. et al. (1992) Biochem. Biophys. Res. Commun.188:1139-1145.) The dehydrogenation reaction converts the NAD analogacetylpyridine adenine dinucleotide (APAD) to the reduced form APADH,which absorbs at 365 nm. A solution of 100 mM Tris-HCl (pH 8), 1 mMAPAD, 0.1% (w/v) Triton X-100, 7.1 units/ml 3-hydroxyacyl-CoAdehydrogenase from pig heart, and sample is mixed in an optical cuvetteand incubated for two minutes at 37° C. Crotonyl-CoA ortrans-2-dodecenoyl-CoA at a final concentration of 100 μM is added tostart the reaction. The change in absorbance at 365 nm is measured usingan ultraviolet spectrophotometer. The amount of HUFA-2 in the sample isproportional to the increase in absorbance at 365 nm at 37° C.

XI. Production of HUFA Specific Antibodies

HUFA substantially purified using PAGE electrophoresis (Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purificationtechniques, is used to immunize rabbits and to produce antibodies usingstandard protocols. The HUFA amino acid sequence is analyzed usingLASERGENE software (DNASTAR Inc.) to determine regions of highimmunogenicity, and a corresponding oligopeptide is synthesized and usedto raise antibodies by means known to those of skill in the art.Selection of appropriate epitopes, such as those near the C-terminus orin hydrophilic regions, is described by Ausubel F. M. et al. (1995 andperiodic supplements) Current Protocols in Molecular Biology, ch. 11,John Wiley & Sons, New York, N.Y. and by others.

Typically, the oligopeptides are 15 residues in length, and aresynthesized using an Applied Biosystems peptide synthesizer usingfmoc-chemistry and coupled to KLH (Sigma, St. Louis, Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinitride ester (MBS), following theprocedure described in Ausubel et al., supra. Rabbits are immunized withthe oligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio-iodinated goat anti-rabbitIgG.

XII. Purification of Naturally Occurring HUFA Using Specific Antibodies

Naturally occurring or recombinant HUFA is substantially purified byimmunoaffinity chromatography using antibodies specific for HUFA. Animmunoaffinity column is constructed by covalently coupling HUFAantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing HUFA are passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof HUFA (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/HUFA binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and HUFAis collected.

XIII. Identification of Molecules Which Interact with HUFA

HUFA or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent. (Bolton et al. (1973) Biochem. J. 133:529.)Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled HUFA, washed, and any wells withlabeled HUFA complex are assayed. Data obtained using differentconcentrations of HUFA are used to calculate values for the number,affinity, and association of HUFA with the candidate molecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 10                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 303 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: BRSTTUT03                                                        (B) CLONE: 1995961                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  Met Ala Ser Trp Ala Lys Gly Arg Ser Tyr - #Leu Ala Pro Gly Leu        Leu                                                                               1               5 - #                 10 - #                 15             - -  Gln Gly Gln Val Ala Ile Val Thr Gly Gly - #Ala Thr Gly Ile Gly Lys                   20     - #             25     - #             30                  - -  Ala Ile Val Lys Glu Leu Leu Glu Leu Gly - #Ser Asn Val Val Ile Ala               35         - #         40         - #         45                      - -  Ser Arg Lys Leu Glu Arg Leu Lys Ser Ala - #Ala Asp Glu Leu Gln Ala           50             - #     55             - #     60                          - -  Asn Leu Pro Pro Thr Lys Gln Ala Arg Val - #Ile Pro Ile Gln Cys Asn       65                 - # 70                 - # 75                 - # 80       - -  Ile Arg Asn Glu Glu Glu Val Asn Asn Leu - #Val Lys Ser Thr Leu Asp                       85 - #                 90 - #                 95              - -  Thr Phe Gly Lys Ile Asn Phe Leu Val Asn - #Asn Gly Gly Gly Gln Phe                   100     - #            105     - #            110                 - -  Leu Ser Pro Ala Glu His Ile Ser Ser Lys - #Gly Trp His Ala Val Leu               115         - #        120         - #        125                     - -  Glu Thr Asn Leu Thr Gly Thr Phe Tyr Met - #Cys Lys Ala Val Tyr Ser           130             - #    135             - #    140                         - -  Ser Trp Met Lys Glu His Gly Gly Ser Ile - #Val Asn Ile Ile Val Pro       145                 - #150                 - #155                 -         #160                                                                             - -  Thr Lys Ala Gly Phe Pro Leu Ala Val His - #Ser Gly Ala Ala Arg        Ala                                                                                              165 - #                170 - #                175            - -  Gly Val Tyr Asn Leu Thr Lys Ser Leu Ala - #Leu Glu Trp Ala Cys Ser                   180     - #            185     - #            190                 - -  Gly Ile Arg Ile Asn Cys Val Ala Pro Gly - #Val Ile Tyr Ser Gln Thr               195         - #        200         - #        205                     - -  Ala Val Glu Asn Tyr Gly Ser Trp Gly Gln - #Ser Phe Phe Glu Gly Ser           210             - #    215             - #    220                         - -  Phe Gln Lys Ile Pro Ala Lys Arg Ile Gly - #Val Pro Glu Glu Val Ser       225                 - #230                 - #235                 -         #240                                                                             - -  Ser Val Val Cys Phe Leu Leu Ser Pro Ala - #Ala Ser Phe Ile Thr        Gly                                                                                              245 - #                250 - #                255            - -  Gln Ser Val Asp Val Asp Gly Gly Arg Ser - #Leu Tyr Thr His Ser Tyr                   260     - #            265     - #            270                 - -  Glu Val Pro Asp His Asp Asn Trp Pro Lys - #Gly Ala Gly Asp Leu Ser               275         - #        280         - #        285                     - -  Val Val Lys Lys Met Lys Glu Thr Phe Lys - #Glu Lys Ala Lys Leu               290             - #    295             - #    300                         - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1210 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: BRSTTUT03                                                        (B) CLONE: 1995961                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  NNCCTGAGAC CCAGAAGGGC CTGCGCTCAG CTGCTCTGGC ACCCCGCTGC - #AGGGATGGC    C    60                                                                         - -  TCCTGGGCTA AGGGCAGGAG CTACCTGGCG CCTGGTTTGC TGCAGGGCCA - #AGTGGCCAT    C   120                                                                         - -  GTCACCGGCG GGGCCACGGG CATCGGAAAA GCCATCGTGA AGGAGCTCCT - #GGAGCTGGG    G   180                                                                         - -  AGTAATGTGG TCATTGCATC CCGTAAGTTG GAGAGATTGA AGTCTGCGGC - #AGATGAACT    G   240                                                                         - -  CAGGCCAACC TACCTCCCAC AAAGCAGGCA CGAGTCATTC CCATACAATG - #CAACATCCG    G   300                                                                         - -  AATGAGGAGG AGGTGAATAA TTTGGTCAAA TCTACCTTAG ATACTTTTGG - #TAAGATCAA    T   360                                                                         - -  TTCTTGGTGA ACAATGGAGG AGGCCAGTTT CTTTCCCCTG CTGAACACAT - #CAGTTCTAA    G   420                                                                         - -  GGATGGCACG CTGTGCTTGA GACCAACCTG ACGGGTACCT TCTACATGTG - #CAAAGCAGT    T   480                                                                         - -  TACAGCTCCT GGATGAAAGA GCATGGAGGA TCTATCGTCA ATATCATTGT - #CCCTACTAA    A   540                                                                         - -  GCTGGATTTC CATTAGCTGT GCATTCTGGA GCTGCAAGAG CAGGTGTTTA - #CAACCTCAC    C   600                                                                         - -  AAATCTTTAG CTTTGGAATG GGCCTGCAGT GGAATACGGA TCAATTGTGT - #TGCCCCTGG    A   660                                                                         - -  GTTATTTATT CCCAGACTGC TGTGGAGAAC TATGGTTCCT GGGGACAAAG - #CTTCTTTGA    A   720                                                                         - -  GGGTCTTTTC AGAAAATCCC CGCTAAACGA ATTGGTGTTC CTGAGGAGGT - #CTCCTCTGT    G   780                                                                         - -  GTCTGCTTCC TACTGTCTCC TGCAGCTTCC TTCATCACTG GACAGTCGGT - #GGATGTGGA    T   840                                                                         - -  GGGGGCCGGA GTCTCTATAC TCACTCGTAT GAGGTACCAG ATCATGACAA - #CTGGCCCAA    G   900                                                                         - -  GGAGCAGGGG ACCTTTCTGT TGTCAAAAAG ATGAAGGAGA CCTTTAAGGA - #GAAAGCTAA    G   960                                                                         - -  CTCTGAGCTG AGGAAACAAG GTGTCCTCCA TCCCCCAGTG CCTTCACATC - #TTGAGGATA    T  1020                                                                         - -  GCTTCTGTAC TTTTTAAAAG CTTATAGTTG GTATGGAAAA CATTTTTCTT - #ATTTTTAAG    T  1080                                                                         - -  GTTATTAATT ATATCTATGG AAAAACTATT CCTGAAATAT ATACAGTCTT - #ATGTCGCAA    T  1140                                                                         - -  CAGAGTCTTT TAACCTATGA TTTAAGAATG TATAAGTAAC AGAGATTAAC - #ATATTTTAA    T  1200                                                                         - -  GACTTTACTC               - #                  - #                      - #      1210                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 301 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: OVARTUT02                                                        (B) CLONE: 2595635                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  Met Ala Lys Ser Leu Leu Lys Thr Ala Ser - #Leu Ser Gly Arg Thr Lys        1               5 - #                 10 - #                 15              - -  Leu Leu His Gln Thr Gly Leu Ser Leu Tyr - #Ser Thr Ser His Gly Phe                   20     - #             25     - #             30                  - -  Tyr Glu Glu Glu Val Lys Lys Thr Leu Gln - #Gln Phe Pro Gly Gly Ser               35         - #         40         - #         45                      - -  Ile Asp Leu Gln Lys Glu Asp Asn Gly Ile - #Gly Ile Leu Thr Leu Asn           50             - #     55             - #     60                          - -  Asn Pro Ser Arg Met Asn Ala Phe Ser Gly - #Val Met Met Leu Gln Leu       65                 - # 70                 - # 75                 - # 80       - -  Leu Glu Lys Val Ile Glu Leu Glu Asn Trp - #Thr Glu Gly Lys Gly Leu                       85 - #                 90 - #                 95              - -  Ile Val Arg Gly Ala Lys Asn Thr Phe Ser - #Ser Gly Ser Asp Leu Asn                   100     - #            105     - #            110                 - -  Ala Val Lys Ser Leu Gly Thr Pro Glu Asp - #Gly Met Ala Val Cys Met               115         - #        120         - #        125                     - -  Phe Met Gln Asn Thr Leu Thr Arg Phe Met - #Arg Leu Pro Leu Ile Ser           130             - #    135             - #    140                         - -  Val Ala Leu Val Gln Gly Trp Ala Leu Gly - #Gly Gly Ala Glu Phe Thr       145                 - #150                 - #155                 -         #160                                                                             - -  Thr Ala Cys Asp Phe Arg Leu Met Thr Pro - #Glu Ser Lys Ile Arg        Phe                                                                                              165 - #                170 - #                175            - -  Val His Lys Glu Met Gly Ile Ile Pro Ser - #Trp Gly Gly Thr Thr Arg                   180     - #            185     - #            190                 - -  Leu Val Glu Ile Ile Gly Ser Arg Gln Ala - #Leu Lys Val Leu Ser Gly               195         - #        200         - #        205                     - -  Ala Leu Lys Leu Asp Ser Lys Asn Ala Leu - #Asn Ile Gly Met Val Glu           210             - #    215             - #    220                         - -  Glu Val Leu Gln Ser Ser Asp Glu Thr Lys - #Ser Leu Glu Glu Ala Gln       225                 - #230                 - #235                 -         #240                                                                             - -  Glu Trp Leu Lys Gln Phe Ile Gln Gly Pro - #Pro Glu Val Ile Arg        Ala                                                                                              245 - #                250 - #                255            - -  Leu Lys Lys Ser Val Cys Ser Gly Arg Glu - #Leu Tyr Leu Glu Glu Ala                   260     - #            265     - #            270                 - -  Leu Gln Asn Glu Arg Asp Leu Leu Gly Thr - #Val Trp Gly Gly Pro Ala               275         - #        280         - #        285                     - -  Asn Leu Glu Ala Ile Ala Lys Lys Gly Lys - #Phe Asn Lys                       290             - #    295             - #    300                         - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2714 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: OVARTUT02                                                        (B) CLONE: 2595635                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -  NAAGCACTTC CTGTCTGCGG CATACAAAAT GTATGGCACG GAATTTTAAG - #CTTACTGAG    C    60                                                                         - -  TTTATAAACA CGTCACATTC ACACATTCAA GACACACACT GGATATTCGG - #ATAAAAACA    A   120                                                                         - -  ACAAACAAAA AACAGGCTAA ATACCCATTC CCCTCAATAA CTTGGATAAG - #ATACCTAAA    A   180                                                                         - -  AAGGTCGACT TCGGTACCTT TCTGTCTTCT CCCCTCTCGC TATTTGCCTA - #CACTGGCTT    C   240                                                                         - -  CTCACCTCCA CTTTTTCTCA CGTTTATCTG AGCGAAAACA AGCACGGTTC - #GGCAGCCTC    C   300                                                                         - -  TTTCCCAGCC CTACCTTTGT GCTGCAAAAG CGAAAATTCA AAAGCCAAGT - #ACAATAGGA    G   360                                                                         - -  ACCGCCCACC CTGGCTCCCT CGTGACACGA GGGAGCGCGA AGCGGAGGGC - #GCCTCGCGG    C   420                                                                         - -  AGGAGCGGGA TTTCCGGGGT CACGGGAACC GGCAGGGGAA CGGGATAAAG - #TTCCTGGAG    A   480                                                                         - -  AAGGAAAGGA GAGCGTGGGA TAGTAAAAGA GAAGACGCGG AGAAGAGGAG - #AGGACCTAC    A   540                                                                         - -  AGAACGGAGG ACAGGGGCGC ACGATGGTCC CGGGGGGAGC GGAAACAAAG - #GCACGCAAA    A   600                                                                         - -  CGGAAAAGCG TGTGTAGGGG AGCGGAAAAG GAAGTCACCA CCGTGGCCTG - #CGACGAAAT    G   660                                                                         - -  GCGAAAAGTC TTTTGAAGAC AGCCTCTCTG TCTGGAAGGA CAAAATTGCT - #ACATCAAAC    A   720                                                                         - -  GGATTGTCAC TTTATAGTAC ATCCCATGGA TTTTATGAGG AAGAAGTGAA - #AAAAACACT    T   780                                                                         - -  CAGCAGTTTC CTGGTGGATC CATTGACCTT CAGAAGGAAG ACAATGGCAT - #TGGCATTCT    T   840                                                                         - -  ACTCTGAACA ATCCAAGTAG AATGAATGCC TTTTCAGGTG TTATGATGCT - #ACAACTTCT    G   900                                                                         - -  GAAAAAGTAA TTGAATTGGA AAATTGGACA GAGGGGAAAG GCCTCATTGT - #CCGTGGGGC    A   960                                                                         - -  AAAAATACTT TCTCTTCAGG ATCTGATCTG AATGCTGTGA AATCACTAGG - #AACTCCAGA    G  1020                                                                         - -  GATGGAATGG CCGTATGCAT GTTCATGCAA AACACCTTAA CAAGATTTAT - #GAGACTTCC    T  1080                                                                         - -  TTAATAAGTG TTGCGCTGGT TCAAGGTTGG GCATTGGGTG GAGGAGCAGA - #ATTTACTAC    A  1140                                                                         - -  GCATGTGATT TCAGGTTAAT GACTCCAGAG AGTAAGATCA GATTCGTCCA - #CAAAGAGAT    G  1200                                                                         - -  GGCATAATAC CAAGCTGGGG TGGCACCACC CGGCTAGTTG AAATAATCGG - #AAGTAGACA    A  1260                                                                         - -  GCTCTCAAAG TGTTGAGTGG GGCCCTTAAA CTGGATTCAA AAAATGCTCT - #AAACATAGG    A  1320                                                                         - -  ATGGTTGAAG AGGTCTTGCA GTCTTCAGAT GAAACTAAAT CTCTAGAAGA - #GGCACAAGA    A  1380                                                                         - -  TGGCTAAAGC AATTCATCCA AGGGCCACCG GAAGTAATTA GAGCTTTGAA - #AAAATCTGT    T  1440                                                                         - -  TGTTCAGGCA GAGAGCTATA TTTGGAGGAA GCATTACAGA ACGAAAGAGA - #TCTTTTAGG    A  1500                                                                         - -  ACAGTTTGGG GTGGGCCTGC AAATTTAGAG GCTATTGCTA AGAAAGGAAA - #ATTTAATAA    A  1560                                                                         - -  TAATTGGTTT TTCGTGTGGA TGTACTCCAA GTAAAGCTCC AGTGACTAAT - #ATGTATAAA    T  1620                                                                         - -  GTTAAATGAT ATTAAATATG AACATCAGAA TTACTTTGAA GGCTACTATT - #AATATGCAG    A  1680                                                                         - -  CTTACTTTTA ATCATTTGAA TATCTGAACT CATTTACCTC ATTTCTTGCC - #AATTACTCA    C  1740                                                                         - -  TTGGGTATTT ACTGCGTAAT CTGGAACATT TAGCTAAAAT ATACACTTTT - #GGCTTAAAA    A  1800                                                                         - -  TTATTGCTGT CAATTCCAAT AATAATTCTT AGCTTATAAC CAAAGAGCAG - #TGTTTAAAA    G  1860                                                                         - -  GAGAGCTTCT ATACAAAACC TATTCCTGGC GTTACTTTTC ATACAATTTT - #TGTTCTGTT    T  1920                                                                         - -  TACCTGGAAA TAATTTACCA AAATAACTGA GTGTTGCTGC TAAAGAACAA - #AAGTGGGGA    G  1980                                                                         - -  GTATCAGGGA ACAAGAAAAC AAGAAAGGGT ATGATCAATC ATTTTCTTCT - #GCTCCAAAC    A  2040                                                                         - -  GCTGGAGTAA AATTCATGGG AAATGGCCCT TCATTTAAAA AAAGATGTAC - #CTCACTACC    C  2100                                                                         - -  ACTACAAATT TGGAACTTTG TTCTTTTCAA TAATTAGTTT TCTATTGTAA - #ATTACCTAC    T  2160                                                                         - -  AAACAGTGGT AGCCATGACA TGGAAAGTCA ACTGATTCTA CAATTGGACA - #TTCATTTGT    G  2220                                                                         - -  TGCCCTGGAA TTTCCAACTA GTAATAAACA ACTACTGTTG ATGTAGTTTT - #AAACCACTT    G  2280                                                                         - -  AAGGGACTCA TGAAGCATCC TGCAACATAA ATTTGCATTT TTACATCAGA - #TTTCTTTTT    T  2340                                                                         - -  TTCCTGAAAA ACAACTAACC TTCTAACAAC TATCTTTCAA AAGTAAATGT - #AATAAAAAT    G  2400                                                                         - -  CACAACATAA AATGTTTATG ATCCCAGCAA TACACTTTTT AAAAAATGTG - #AAAGTCAAA    G  2460                                                                         - -  AATTAAGTTC TAGTTCTGAC TCATCACAAG AGGTCAAAAG TATTTGCTAC - #TGTAACATT    C  2520                                                                         - -  AATTCACATT TGAGAATCAT GGTAAAAATA ACTTGTATTT GCCTTACCAT - #CATGATCCT    A  2580                                                                         - -  CTGTTGAGTT AGGAAAATAT GGTTAGACAG ACTCACATTA CTTTTTTTCA - #GAGGTAAAC    T  2640                                                                         - -  CTAGATTACT GTGTCAACCC AATACTATTT GGCCATAGAT GTAAAAACTA - #CCAAATAAA    A  2700                                                                         - -  GTGGATTTTG TGTC             - #                  - #                      - #   2714                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 295 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 730864                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - -  Met Asp Thr Met Asn Thr Ala Asn Thr Leu - #Asp Gly Lys Phe Val Thr        1               5 - #                 10 - #                 15              - -  Glu Gly Ser Trp Arg Pro Asp Leu Phe Lys - #Gly Lys Val Ala Phe Val                   20     - #             25     - #             30                  - -  Thr Gly Gly Ala Gly Thr Ile Cys Arg Val - #Gln Thr Glu Ala Leu Val               35         - #         40         - #         45                      - -  Leu Leu Gly Cys Lys Ala Ala Ile Val Gly - #Arg Asp Gln Glu Arg Thr           50             - #     55             - #     60                          - -  Glu Gln Ala Ala Lys Gly Ile Ser Gln Leu - #Ala Lys Asp Lys Asp Ala       65                 - # 70                 - # 75                 - # 80       - -  Val Leu Ala Ile Ala Asn Val Asp Val Arg - #Asn Phe Glu Gln Val Glu                       85 - #                 90 - #                 95              - -  Asn Ala Val Lys Lys Thr Val Glu Lys Phe - #Gly Lys Ile Asp Phe Val                   100     - #            105     - #            110                 - -  Ile Ala Gly Ala Ala Gly Asn Phe Val Cys - #Asp Phe Ala Asn Leu Ser               115         - #        120         - #        125                     - -  Pro Asn Ala Phe Lys Ser Val Val Asp Ile - #Asp Leu Leu Gly Ser Phe           130             - #    135             - #    140                         - -  Asn Thr Ala Lys Ala Cys Leu Lys Glu Leu - #Lys Lys Ser Lys Gly Ser       145                 - #150                 - #155                 -         #160                                                                             - -  Ile Leu Phe Val Ser Ala Thr Phe His Tyr - #Tyr Gly Val Pro Phe        Gln                                                                                              165 - #                170 - #                175            - -  Gly His Val Gly Ala Ala Lys Ala Gly Ile - #Asp Ala Leu Ala Lys Asn                   180     - #            185     - #            190                 - -  Leu Ala Val Glu Leu Gly Pro Leu Gly Ile - #Arg Ser Asn Cys Ile Ala               195         - #        200         - #        205                     - -  Pro Gly Ala Ile Asp Asn Thr Glu Gly Leu - #Lys Arg Leu Ala Gly Lys           210             - #    215             - #    220                         - -  Lys Tyr Lys Glu Lys Ala Leu Ala Lys Ile - #Pro Leu Gln Arg Leu Gly       225                 - #230                 - #235                 -         #240                                                                             - -  Ser Thr Arg Asp Ile Ala Glu Ser Thr Val - #Tyr Ile Phe Ser Pro        Ala                                                                                              245 - #                250 - #                255            - -  Ala Ser Tyr Val Thr Gly Thr Val Leu Val - #Val Asp Gly Gly Met Trp                   260     - #            265     - #            270                 - -  His Leu Gly Thr Tyr Phe Gly His Glu Leu - #Tyr Pro Glu Ala Leu Ile               275         - #        280         - #        285                     - -  Lys Ser Met Thr Ser Lys Leu                                                  290             - #    295                                                - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 335 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 602703                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - -  Met Lys Leu Pro Ala Arg Val Phe Phe Thr - #Leu Gly Ser Arg Leu Pro        1               5 - #                 10 - #                 15              - -  Cys Gly Leu Ala Pro Arg Arg Phe Phe Ser - #Tyr Gly Thr Lys Ile Leu                   20     - #             25     - #             30                  - -  Tyr Gln Asn Thr Glu Ala Leu Gln Ser Lys - #Phe Phe Ser Pro Leu Gln               35         - #         40         - #         45                      - -  Lys Ala Met Leu Pro Pro Asn Ser Phe Gln - #Gly Lys Val Ala Phe Ile           50             - #     55             - #     60                          - -  Thr Gly Gly Gly Thr Gly Leu Gly Lys Gly - #Met Thr Thr Leu Leu Ser       65                 - # 70                 - # 75                 - # 80       - -  Ser Leu Gly Ala Gln Cys Val Ile Ala Ser - #Arg Lys Met Asp Val Leu                       85 - #                 90 - #                 95              - -  Lys Ala Thr Ala Glu Gln Ile Ser Ser Gln - #Thr Gly Asn Lys Val His                   100     - #            105     - #            110                 - -  Ala Ile Gln Cys Asp Val Arg Asp Pro Asp - #Met Val Gln Asn Thr Val               115         - #        120         - #        125                     - -  Ser Glu Leu Ile Lys Val Ala Gly His Pro - #Asn Ile Val Ile Asn Asn           130             - #    135             - #    140                         - -  Ala Ala Gly Asn Phe Ile Ser Pro Thr Glu - #Arg Leu Ser Pro Asn Ala       145                 - #150                 - #155                 -         #160                                                                             - -  Trp Lys Thr Ile Thr Asp Ile Val Leu Asn - #Gly Thr Ala Phe Val        Thr                                                                                              165 - #                170 - #                175            - -  Leu Glu Ile Gly Lys Gln Leu Ile Lys Ala - #Gln Lys Gly Ala Ala Phe                   180     - #            185     - #            190                 - -  Leu Ser Ile Thr Thr Ile Tyr Ala Glu Thr - #Gly Ser Gly Phe Val Val               195         - #        200         - #        205                     - -  Pro Ser Ala Ser Ala Lys Ala Gly Val Glu - #Ala Met Ser Lys Ser Leu           210             - #    215             - #    220                         - -  Ala Ala Glu Trp Gly Lys Tyr Gly Met Arg - #Phe Asn Val Ile Gln Pro       225                 - #230                 - #235                 -         #240                                                                             - -  Gly Pro Ile Lys Thr Lys Gly Ala Phe Ser - #Arg Leu Asp Pro Thr        Gly                                                                                              245 - #                250 - #                255            - -  Thr Phe Glu Lys Glu Met Ile Gly Arg Ile - #Pro Cys Gly Arg Leu Gly                   260     - #            265     - #            270                 - -  Thr Val Glu Glu Leu Ala Asn Leu Ala Ala - #Phe Leu Cys Ser Asp Tyr               275         - #        280         - #        285                     - -  Ala Ser Trp Ile Asn Gly Ala Val Ile Lys - #Phe Asp Gly Gly Glu Glu           290             - #    295             - #    300                         - -  Val Leu Ile Ser Gly Glu Phe Asn Asp Leu - #Arg Lys Val Thr Lys Glu       305                 - #310                 - #315                 -         #320                                                                             - -  Gln Trp Asp Thr Ile Glu Glu Leu Ile Arg - #Lys Thr Lys Gly Ser                          325 - #                330 - #                335             - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 335 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 111287                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - -  Met Ala Leu Leu Ala Arg Ala Phe Phe Ala - #Gly Val Ser Arg Leu Pro        1               5 - #                 10 - #                 15              - -  Cys Asp Pro Gly Pro Gln Arg Phe Phe Ser - #Phe Gly Thr Lys Thr Leu                   20     - #             25     - #             30                  - -  Tyr Gln Ser Ile Asp Ala Pro Gln Ser Lys - #Phe Phe Pro Pro Ile Leu               35         - #         40         - #         45                      - -  Lys Pro Met Leu Pro Pro Asn Ala Phe Gln - #Gly Lys Val Ala Phe Ile           50             - #     55             - #     60                          - -  Thr Gly Gly Gly Thr Gly Leu Gly Lys Ala - #Met Thr Thr Phe Leu Ser       65                 - # 70                 - # 75                 - # 80       - -  Ser Leu Gly Ala Gln Cys Val Ile Ala Ser - #Arg Asn Ile Asp Val Leu                       85 - #                 90 - #                 95              - -  Lys Ala Thr Ala Glu Glu Ile Thr Ser Lys - #Thr Gly Asn Lys Val Tyr                   100     - #            105     - #            110                 - -  Ala Ile Arg Cys Asp Val Arg Asp Pro Asp - #Met Val His Asn Thr Val               115         - #        120         - #        125                     - -  Leu Glu Leu Ile Lys Val Ala Gly His Pro - #Asp Val Val Ile Asn Asn           130             - #    135             - #    140                         - -  Ala Ala Gly Asn Phe Ile Ser Pro Ser Glu - #Arg Leu Ser Pro Asn Gly       145                 - #150                 - #155                 -         #160                                                                             - -  Trp Lys Thr Ile Thr Asp Ile Val Leu Asn - #Gly Thr Ala Tyr Val        Thr                                                                                              165 - #                170 - #                175            - -  Ile Glu Ile Gly Lys Gln Leu Ile Lys Ala - #Gln Lys Gly Ala Ala Phe                   180     - #            185     - #            190                 - -  Leu Ala Ile Thr Thr Ile Tyr Ala Glu Ser - #Gly Ser Gly Phe Val Met               195         - #        200         - #        205                     - -  Pro Ser Ser Ser Ala Lys Ser Gly Val Glu - #Ala Met Asn Lys Ser Leu           210             - #    215             - #    220                         - -  Ala Ala Glu Trp Gly Arg Tyr Gly Met Arg - #Phe Asn Ile Ile Gln Pro       225                 - #230                 - #235                 -         #240                                                                             - -  Gly Pro Ile Lys Thr Lys Gly Ala Phe Ser - #Arg Leu Asp Pro Thr        Gly                                                                                              245 - #                250 - #                255            - -  Lys Phe Glu Lys Asp Met Ile Glu Arg Ile - #Pro Cys Gly Arg Leu Gly                   260     - #            265     - #            270                 - -  Thr Val Glu Glu Leu Ala Asn Leu Ala Thr - #Phe Leu Cys Ser Asp Tyr               275         - #        280         - #        285                     - -  Ala Ser Trp Ile Asn Gly Ala Val Ile Arg - #Phe Asp Gly Gly Glu Glu           290             - #    295             - #    300                         - -  Val Phe Leu Ser Gly Glu Phe Asn Ser Leu - #Lys Lys Val Thr Lys Glu       305                 - #310                 - #315                 -         #320                                                                             - -  Glu Trp Asp Val Ile Glu Gly Leu Ile Arg - #Lys Thr Lys Gly Ser                          325 - #                330 - #                335             - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 339 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 780241                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - -  Met Ala Ala Ala Val Ala Ala Ala Pro Gly - #Ala Leu Gly Ser Leu His        1               5 - #                 10 - #                 15              - -  Ala Gly Gly Ala Arg Leu Val Ala Ala Cys - #Ser Ala Trp Leu Cys Pro                   20     - #             25     - #             30                  - -  Gly Leu Arg Leu Pro Gly Ser Leu Ala Gly - #Arg Arg Ala Gly Pro Ala               35         - #         40         - #         45                      - -  Ile Trp Ala Gln Gly Trp Val Pro Ala Ala - #Gly Gly Pro Ala Pro Lys           50             - #     55             - #     60                          - -  Arg Gly Tyr Ser Ser Glu Met Lys Thr Glu - #Asp Glu Leu Arg Val Arg       65                 - # 70                 - # 75                 - # 80       - -  His Leu Glu Glu Glu Asn Arg Gly Ile Val - #Val Leu Gly Ile Asn Arg                       85 - #                 90 - #                 95              - -  Ala Tyr Gly Lys Asn Ser Leu Ser Lys Asn - #Leu Ile Lys Met Leu Ser                   100     - #            105     - #            110                 - -  Lys Ala Val Asp Ala Leu Lys Ser Asp Lys - #Lys Val Arg Thr Ile Ile               115         - #        120         - #        125                     - -  Ile Arg Ser Glu Val Pro Gly Ile Phe Cys - #Ala Gly Ala Asp Leu Lys           130             - #    135             - #    140                         - -  Glu Arg Ala Lys Met Ser Ser Ser Glu Val - #Gly Pro Phe Val Ser Lys       145                 - #150                 - #155                 -         #160                                                                             - -  Ile Arg Ala Val Ile Asn Asp Ile Ala Asn - #Leu Pro Val Pro Thr        Ile                                                                                              165 - #                170 - #                175            - -  Ala Ala Ile Asp Gly Leu Ala Leu Gly Gly - #Gly Leu Glu Leu Ala Leu                   180     - #            185     - #            190                 - -  Ala Cys Asp Ile Arg Val Ala Ala Ser Ser - #Ala Lys Met Gly Leu Val               195         - #        200         - #        205                     - -  Glu Thr Lys Leu Ala Ile Ile Pro Gly Gly - #Gly Gly Thr Gln Arg Leu           210             - #    215             - #    220                         - -  Pro Arg Ala Ile Gly Met Ser Leu Ala Lys - #Glu Leu Ile Phe Ser Ala       225                 - #230                 - #235                 -         #240                                                                             - -  Arg Val Leu Asp Gly Lys Glu Ala Lys Ala - #Val Gly Leu Ile Ser        His                                                                                              245 - #                250 - #                255            - -  Val Leu Glu Gln Asn Gln Glu Gly Asp Ala - #Ala Tyr Arg Lys Ala Leu                   260     - #            265     - #            270                 - -  Asp Leu Ala Arg Glu Phe Leu Pro Gln Gly - #Pro Val Ala Met Arg Val               275         - #        280         - #        285                     - -  Ala Lys Leu Ala Ile Asn Gln Gly Met Glu - #Val Asp Leu Val Thr Gly           290             - #    295             - #    300                         - -  Leu Ala Ile Glu Glu Ala Cys Tyr Ala Gln - #Thr Ile Pro Thr Lys Asp       305                 - #310                 - #315                 -         #320                                                                             - -  Arg Leu Glu Gly Leu Leu Ala Phe Lys Glu - #Lys Arg Pro Pro Arg        Tyr                                                                                              325 - #                330 - #                335            - -  Lys Gly Glu                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 290 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 1922287                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - -  Met Ala Ala Leu Arg Val Leu Leu Ser Cys - #Ala Arg Gly Pro Leu Arg        1               5 - #                 10 - #                 15              - -  Pro Pro Val Arg Cys Pro Ala Trp Arg Pro - #Phe Ala Ser Gly Ala Asn                   20     - #             25     - #             30                  - -  Phe Glu Tyr Ile Ile Ala Glu Lys Arg Gly - #Lys Asn Asn Thr Val Gly               35         - #         40         - #         45                      - -  Leu Ile Gln Leu Asn Arg Pro Lys Ala Leu - #Asn Ala Leu Cys Asp Gly           50             - #     55             - #     60                          - -  Leu Ile Asp Glu Leu Asn Gln Ala Leu Lys - #Ile Phe Glu Glu Asp Pro       65                 - # 70                 - # 75                 - # 80       - -  Ala Val Gly Ala Ile Val Leu Thr Gly Gly - #Asp Lys Ala Phe Ala Ala                       85 - #                 90 - #                 95              - -  Gly Ala Asp Ile Lys Glu Met Gln Asn Leu - #Ser Phe Gln Asp Cys Tyr                   100     - #            105     - #            110                 - -  Ser Ser Lys Phe Leu Lys His Trp Asp His - #Leu Thr Gln Val Lys Lys               115         - #        120         - #        125                     - -  Pro Val Ile Ala Ala Val Asn Gly Tyr Ala - #Phe Gly Gly Gly Cys Glu           130             - #    135             - #    140                         - -  Leu Ala Met Met Cys Asp Ile Ile Tyr Ala - #Gly Glu Lys Ala Gln Phe       145                 - #150                 - #155                 -         #160                                                                             - -  Ala Gln Pro Glu Ile Leu Ile Gly Thr Ile - #Pro Gly Ala Gly Gly        Thr                                                                                              165 - #                170 - #                175            - -  Gln Arg Leu Thr Arg Ala Val Gly Lys Ser - #Leu Ala Met Glu Met Val                   180     - #            185     - #            190                 - -  Leu Thr Gly Asp Arg Ile Ser Ala Gln Asp - #Ala Lys Gln Ala Gly Leu               195         - #        200         - #        205                     - -  Val Ser Lys Ile Cys Pro Val Glu Thr Leu - #Val Glu Glu Ala Ile Gln           210             - #    215             - #    220                         - -  Cys Ala Glu Lys Ile Ala Ser Asn Ser Lys - #Ile Val Val Ala Met Ala       225                 - #230                 - #235                 -         #240                                                                             - -  Lys Glu Ser Val Asn Ala Ala Phe Glu Met - #Thr Leu Thr Glu Gly        Ser                                                                                              245 - #                250 - #                255            - -  Lys Leu Glu Lys Lys Leu Phe Tyr Ser Thr - #Phe Ala Thr Asp Asp Arg                   260     - #            265     - #            270                 - -  Lys Glu Gly Met Thr Ala Phe Val Glu Lys - #Arg Lys Ala Asn Phe Lys               275         - #        280         - #        285                     - -  Asp Gln                                                                      290                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 328 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 564065                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - -  Met Ala Ala Gly Ile Val Ala Ser Arg Arg - #Leu Arg Asp Leu Leu Thr        1               5 - #                 10 - #                 15              - -  Arg Arg Leu Thr Gly Ser Asn Tyr Pro Gly - #Leu Ser Ile Ser Leu Arg                   20     - #             25     - #             30                  - -  Leu Thr Gly Ser Ser Ala Gln Glu Glu Ala - #Ser Gly Val Ala Leu Gly               35         - #         40         - #         45                      - -  Glu Ala Pro Asp His Ser Tyr Glu Ser Leu - #Arg Val Thr Ser Ala Gln           50             - #     55             - #     60                          - -  Lys His Val Leu His Val Gln Leu Asn Arg - #Pro Asn Lys Arg Asn Ala       65                 - # 70                 - # 75                 - # 80       - -  Met Asn Lys Val Phe Trp Arg Glu Met Val - #Glu Cys Phe Asn Lys Ile                       85 - #                 90 - #                 95              - -  Ser Arg Asp Ala Asp Cys Arg Ala Val Val - #Ile Ser Gly Ala Gly Lys                   100     - #            105     - #            110                 - -  Met Phe Thr Ala Gly Ile Asp Leu Met Asp - #Met Ala Ser Asp Ile Leu               115         - #        120         - #        125                     - -  Gln Pro Lys Gly Asp Asp Val Ala Arg Ile - #Ser Trp Tyr Leu Arg Asp           130             - #    135             - #    140                         - -  Ile Ile Thr Arg Tyr Gln Glu Thr Phe Asn - #Val Ile Glu Arg Cys Pro       145                 - #150                 - #155                 -         #160                                                                             - -  Lys Pro Val Ile Ala Ala Val His Gly Gly - #Cys Ile Gly Gly Gly        Val                                                                                              165 - #                170 - #                175            - -  Asp Leu Val Thr Ala Cys Asp Ile Arg Tyr - #Cys Ala Gln Asp Ala Phe                   180     - #            185     - #            190                 - -  Phe Gln Val Lys Glu Val Asp Val Gly Leu - #Ala Ala Asp Val Gly Thr               195         - #        200         - #        205                     - -  Leu Glu Arg Leu Pro Lys Val Ile Gly Asn - #Gln Ser Leu Val Asn Glu           210             - #    215             - #    220                         - -  Leu Ala Phe Thr Ala His Lys Met Met Ala - #Asp Glu Ala Leu Asp Ser       225                 - #230                 - #235                 -         #240                                                                             - -  Gly Leu Val Ser Arg Val Phe Pro Asp Lys - #Glu Val Met Leu Asp        Ala                                                                                              245 - #                250 - #                255            - -  Ala Leu Pro Leu Ala Pro Glu Ile Ser Ser - #Lys Thr Thr Val Leu Val                   260     - #            265     - #            270                 - -  Gln Ser Thr Lys Val Asn Leu Leu Tyr Ser - #Arg Asp His Ser Val Ala               275         - #        280         - #        285                     - -  Glu Ser Leu Asn Tyr Val Ala Ser Trp Asn - #Met Ser Met Leu Gln Thr           290             - #    295             - #    300                         - -  Gln Asp Leu Val Lys Ser Val Gln Pro Thr - #Thr Glu Asn Lys Glu Leu       305                 - #310                 - #315                 -         #320                                                                             - -  Lys Thr Val Thr Phe Ser Lys Leu                                                          325                                                        __________________________________________________________________________

What is claimed is:
 1. An isolated and purified polynucleotide sequenceencoding a polypeptide comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 1 and SEQ ID NO:3.
 2. A compositioncomprising the polynucleotide sequence of claim
 1. 3. An isolated andpurified polynucleotide sequence which hybridizes under stringentconditions of 35° C. in 35% formamide to the polynucleotide sequence ofclaim
 1. 4. An isolated and purified polynucleotide sequence which iscomplementary to he polynucleotide sequence of claim
 1. 5. An isolatedand purified polynucleotide sequence comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:2 and SEQ IDNO:4.
 6. An isolated and purified polynucleotide sequence which iscomplementary to the polynucleotide sequence of claim
 5. 7. Anexpression vector containing the polynucleotide sequence of claim
 1. 8.A host cell containing the expression vector of claim
 7. 9. A method forproducing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3, the method comprising the steps of:a) culturing thehost cell of claim 8 under conditions suitable for the expression of thepolypeptide; and b) recovering the polypeptide from the host cellculture.
 10. A method for detecting a polynucleotide encoding HUFA in abiological sample containing nucleic acids, the method comprising thesteps of:(a) hybridizing the polynucleotide of claim 4 to at least oneof the nucleic acids in the biological sample, thereby forming ahybridization complex; and (b) detecting the hybridization complex,wherein the presence of the hybridization complex correlates with thepresence of a polynucleotide encoding HUFA in the biological sample. 11.The method of claim 10 wherein the nucleic acids of the biologicalsample are amplified by the polymerase chain reaction prior tohybridization.